Immunohistochemical profiling of the ultimobranchial remnants in the rat postnatal thyroid gland

Ultimobranchial (UB) remnants are a constant presence in the thyroid throughout rat postnatal life; however, the difficulty in identifying the most immature forms from the surrounding thyroid tissue prompted us to search for a specific marker. With that objective, we applied a panel of antibodies reported to be specific for their human counterpart, solid cell nests (SCNs), using double immunohistochemistry and immunofluorescence. Our results demonstrated that cytokeratin 34βE12 and p63 are highly sensitive markers for the immunohistologic screening of UB‐remnants, independently of their maturity or size. Furthermore, rat UB‐follicles (UBFs) coincided with human SCNs in the immunohistochemical pattern exhibited by both antigens. In contrast, the pattern displayed for calcitonin and thyroglobulin differs considerably but confirm the hypothesis that rat UB‐cells can differentiate into both types of thyroid endocrine cells. This hypothesis agrees with recent findings that thyroid C‐cells share an endodermic origin with follicular cells in rodents. We suggest that the persistence of p63‐positive undifferentiated cells in UB‐remnants may constitute a reservoir of basal/stem cells that persist beyond embryogenesis from which, in certain unknown conditions, differentiated thyroid cells or even unusual tumors may arise.     

Solid cell nests of the human thyroid in early stages of postnatal life. Systematic autopsy study

The anatomical position and prevalence of solid cell nests (SCN) of the thyroid in early stages of postnatal life have not yet been clearly determined. In order to find out about these unsettled questions a systematic search of these ultimobranchial nests from 92 autopsied thyroids from neonates, children and young adults was undertaken. SCN were present in 61% of the patients; they were mainly located in the middle third of the lateral thyroid lobes, and placed along a central to paracentral and slightly dorsal longitudinal axis. These findings, as compared with our previous observations made in older adult thyroids, further demonstrate that there exist a constant anatomical position and prevalence of SCN in postnatal life. The significantly higher frequency of SCN in males (68%) than in females (48%) (p less than 0.01) found in a study that was further extended to 192 thyroids at early and late stages of postnatal life, is a question that requires further investigation.     

Cellular Requirements of Suprachiasmatic Nucleus Transplants for Restoration of Orcadian Rhythm

Fetal neurografts containing the suprachiasmatic nucleus (SCN) can restore the circadian locomotor and drinking rhythm of SCN-lesioned (SCNX) rat and hamster. This functional outcome finally proves that the endogenous biological clock autonomously resides in the SCN. Observations on the cellular requirements of the “new” SCN for restoration of the arrhythmic SCNX animals have led to some new insights and confirmed findings from other studies. A critical mass of SCN neurons appeared necessary for functional effects, whereas the temporal profile of reinstatement of rhythm correlated with the delayed maturation of the grafted SCN. Cytoarchitectoni-cally, the grafted SCN does not seem to develop normally for all anatomical aspects. Complementary clusters of vasoactive intestinal polypeptide(VIP)-and vasopressin(VP)ergic neurons are formed, but somatostatin(SOM)ergic neurons do not always “join” this group, as is normally seen in situ. Nevertheless, these new SCNs can restore the ablated functions. As the period length of restored rhythms tends to vary, it might be that the grafted SCN underwent an altered or impaired maturation that resulted in a different setting of its clock mechanism. A prominent role of VIPergic neurons seems indicated by their presence in all functional grafts, but, although they may be required, these cells do not appear to be a sufficient condition for restoration of rhythm. Many grafts exhibit the presence of VIPergic cells without counteracting the arrhythmia, whereas VP- and SOMergic SCN neurons are usually present as well. Findings with VP-deficient Brattleboro rat grafts indicated that VP is not the primary obligatory signal of circadian activity. It is argued that perhaps the role of SOMergic neurons in the clock function of the (grafted) SCN has been insufficiently considered. However, one should keep in mind that the peptides of the various types of SCN neurons may function only as cofactors, mutually modulating molecular or bioelectrical cellular activities within the nucleus or the message of the main transmitter γ-aminobutyric acid.     

Mixed follicles of the human thyroid gland

Mixed follicles are structures composed of squamous-like and follicular epithelia. Little attention has been generally paid to these peculiar follicles of the human thyroid; thus the aim of the present study was to investigate their prevalence and biological properties by means of systematic autopsy, histochemical and immunohistochemical surveys. Mixed follicles were found to be present in 54% and 81% of the patients with solid cell nests, as well as in 50% and 77% of the total number of the ultimobranchial nests, when one or two histological samples from each solid cell nest were examined, respectively. The follicular lumen of mixed follicles usually contained an eosinophilic and PAS-positive colloid-like material, although in 22% of the cases acid mucins sometimes intermixed with PAS-positive granular material and cell debris were also present within lumina. Follicular cells lining mixed follicles basically did not stain positively for calcitonin. The results indicate that mixed follicles are not rarely found in the human thyroid. The presence of intraluminal mucins and cell debris and the absence of calcitonin-containing cells in the follicular epithelium lining these peculiar follicles suggest that at least some thyroid follicular cells could originate from ultimobranchial tissue.     

Thyroid follicles with acid mucins in man: A second kind of follicles?

Summary Human thyroid follicles containing acid mucins have been regarded as a very rare finding and their significance has not yet been clarified. Therefore, a systematic anatomical, histochemical and immunohistochemical survey for the presence of such follicles in human thyroids was undertaken.Follicles with Alcian blue-positive acid mucins were practically confined to the 18% of sections that also contained ultimobranchial solid cell nests. Immunohistochemistry revealed that these follicles were mostly composed of and/or related to the presence of numerous calcitonin-immunoreactive cells, sometimes intermixed with occasional alcianophilic mucinous cells. These findings, with histometrical studies, demonstrate that there exists a relationship between mucinous C cell complexes and mucin/C cell-containing solid cell nests. The finding of calcitonin immunoreactivity in very occasional groups of cells with mucinous changes further suggests that at least some human follicular cells originate in ultimobranchial tissue.     

The relation between solid cell nests and C cells of the thyroid gland

Summary Thyroid tissue of 300 routine autopsies was processed in a standardized manner. So-called solid cell nests (SCN) were found in 21 patients (7 %). These cases were investigated carefully by serial step sectioning. In order to explore the correlation of SCN to the C-cell system, the sections were stained by silver impregnation and the immunoperoxidase method. Morphometric analyses revealed a significant increase in the density of C cells in the proximity of the SCN. With progressive distance from the SCN, the C-cell density decreased and reached normal values. In 30 % of the cases argyrophilic and calcitonin-positive cells were found lying within the SCN. Occasionally, mixed follicles could be discerned: These were lined on the one side by a multilayered squamous epithelium, on the other side by normal monolayered cubic follicular epithelium, and contained a peculiar granular material. In one case, SCN were associated with intrathyroid portions of the parathyroids and adult adipose tissue, in a second case with adipose tissue only. Most probably SCN are vestiges of the ultimobranchial body and should be interpreted as such, despite the fact that other authors have expressed different views. The lack of disturbances in the calcium metabolism of the patients and the absence of medullary carcinoma in their family histories led us to interpret locally confined C-cell hyperplasia not as reactive nor premalignant, but rather as normal.     

鲫鱼后鳃体结构与功能的研究

本文对鲫鱼后鳃体的形态结构及其功能进行了初步研究。结果表明,鲫鱼后鳃体是一种内分泌腺体。腺体由滤泡组成,滤泡上皮包括二种颗粒细胞(亮细胞和暗细胞)和一种支持细胞。对不同性腺期的鲫鱼后鳃体的观察表明,后鳃体滤泡细胞随性腺发育增加高度,滤泡增生,腺体体积增大。而性腺成熟后期,后鳃体滤泡上皮有崩溃现象。揭示后鳃体可能与性腺发育有关。用免疫组织化学的非标记过氧化物酶技术对鲫鱼后鳃体进行激素降钙素的定性定位研究,观察到滤泡上皮的颗粒细胞发生阳性反应,由此证明颗粒细胞可分泌降钙素。     

长蛸生殖系统的形态学与组织学观察

运用解剖学和组织学方法对长蛸(Octopus variabilis)生殖系统的形态结构进行了研究.结果表明,长蛸雌雄异体并异形,雄性右侧第三腕茎化.长蛸雌性生殖系统由一个卵巢、成对的输卵管及输卵管腺组成.卵巢壁上发出一条线状具分支的生殖索,米粒状的滤泡以卵柄连接至生殖索上.每个滤泡是由单层滤泡细胞围绕着一个卵母细胞构成.输卵管形成丰富的纵行褶皱,黏膜上皮具有纤毛.输卵管腺含有两种类型腺细胞.雄性生殖系统包括精巢、输精管前段、储精囊、摄护腺、盲囊、输精管后段和精荚囊.精巢内部被结缔组织分隔成许多精小叶,精原细胞由小叶壁中的生殖上皮产生,并向小叶腔中逐步分化成精子.输精管前段、盲囊和摄护腺所分泌的黏液物质共同参与精荚的形成.储精囊和输精管后段形成较多的纵行褶皱,输精管后段上皮游离面的纤毛可运输生殖细胞.精荚囊的作用则是贮存精荚,囊壁中的平滑肌利于长蛸交配时精荚的排出.     

Ovary cord structure and oogenesis in Hirudo medicinalis and Haemopis sanguisuga (Clitellata, Annelida): remarks on different ovaries organization in Hirudinea

In Hirudo medicinalis and Haemopis sanguisuga, two convoluted ovary cords are found within each ovary. Each ovary cord is a polarized structure composed of germ cells (oogonia, developing oocytes, nurse cells) and somatic cells (apical cell, follicular cells). One end of the ovary cord is club-shaped and comprises one huge apical cell, numerous oogonia, and small cysts (clusters) of interconnected germ cells. The main part of the cord contains fully developed cysts composed of numerous nurse cells connected via intercellular bridges with the cytophore, which in turn is connected by a cytoplasmic bridge with the growing oocyte. The opposite end of the cord degenerates. Cord integrity is ensured by flattened follicular cells enveloping the cord; moreover, inside the cord, some follicular cells (internal follicular cells) are distributed among germ cells. As oogenesis progresses, the growing oocytes gradually protrude into the ovary lumen; as a result, fully developed oocytes arrested in meiotic metaphase I float freely in the ovary lumen. This paper describes the successive stages of oogenesis of H. medicinalis in detail. Ovary organization in Hirudinea was classified within four different types: non-polarized ovary cords were found in glossiphoniids, egg follicles were described in piscicolids, ovarian bodies were found characteristic for erpobdellids, and polarized ovary cords in hirudiniforms. Ovaries with polarized structures equipped with apical cell (i.e. polarized ovary cords and ovarian bodies) (as found in arhynchobdellids) are considered as primary for Hirudinea while non-polarized ovary cords and the occurrence of egg follicles (rhynchobdellids) represent derived condition.     

The significance of paravacuolar granules of the thyroid. A histologic, cytologic and ultrastructural study

The presence of the so-called "paravacuolar granules" in thyroid follicular cells has been associated with increased metabolic activity of the gland, regressive changes, degeneration, phagocytic activity and benign papillary hyperplasia. During the course of a review of the intraoperative cytologic preparations and corresponding histologic sections from 73 thyroid cases, the presence of granules within follicular cells was noted in 25 cases (18 adenomatous or colloid goiters, 3 follicular adenomas, 2 papillary carcinomas, 1 follicular carcinoma and in thyroid tissue surrounding a follicular adenoma in 1 case). Histochemical and ultrastructural studies showed the granules to consist of lysosomes containing hemosiderin or lipofuscin pigments. These findings indicate that the presence of paravacuolar granules in thyroid cells is a common nonspecific finding that simply reflects: (1) the erythrophagocytic capability of the follicular epithelial cells, which results in the accumulation of iron within lysosomes, and (2) the accumulation of lipofuscin pigments within lysosomes as a result of degradation of endogenous cellular material.     

Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney

In search of guiding principles involved in the branching of epithelial tubes in the developing kidney, we analyzed branching of the ureteric bud (UB) in whole kidney culture as well as in isolated UB culture independent of mesenchyme but in the presence of mesenchymally derived soluble factors. Microinjection of the UB lumen (both in the isolated UB and in the whole kidney) with fluorescently labeled dextran sulfate demonstrated that branching occurred via smooth tubular epithelial outpouches with a lumen continuous with that of the original structure. Epithelial cells within these outpouches cells were wedge-shaped with actin, myosin-2 and ezrin localized to the luminal side, raising the possibility of a "purse-string" mechanism. Electron microscopy and decoration of heparan sulfates with biotinylated FGF2 revealed that the basolateral surface of the cells remained intact, without the type of cytoplasmic extensions (invadopodia) that are seen in three-dimensional MDCK, mIMCD, and UB cell culture models of branching tubulogenesis. Several growth factor receptors (i.e., FGFR1, FGFR2, c-Ret) and metalloproteases (i.e., MT1-MMP) were localized toward branching UB tips. A large survey of markers revealed the ER chaperone BiP to be highly expressed at UB tips, which, by electron microscopy, are enriched in rough endoplasmic reticulum and Golgi, supporting high activity in the synthesis of transmembrane and secretory proteins at UB tips. After early diffuse proliferation, proliferating and mitotic cells were mostly found within the branching ampullae, whereas apoptotic cells were mostly found in stalks. Gene array experiments, together with protein expression analysis by immunoblotting, revealed a differential spatiotemporal distribution of several proteins associated with epithelial maturation and polarization, including intercellular junctional proteins (e.g., ZO-1, claudin-3, E-cadherin) and the subapical cytoskeletal/microvillar protein ezrin. In addition, Ksp-cadherin was found at UB ampullary cells next to developing outpouches, suggesting a role in epithelial-mesenchymal interactions. These data from the isolated UB culture system support a model where UB branching occurs through outpouching possibly mediated by wedge-shaped cells created through an apical cytoskeletal purse-string mechanism. Additional potential mechanisms include (1) differential localization of growth factor receptors and metalloproteases at tips relative to stalks; (2) creation of a secretory epithelium, in part manifested by increased expression of the ER chaperone BiP, at tips relative to stalks; (3) after initial diffuse proliferation, coexistence of a balance of proliferation vs. apoptosis favoring tip growth with a very different balance in elongating stalks; and (4) differential maturation of the tight and adherens junctions as the structures develop. Because, without mesenchyme, both lateral and bifid branching occurs (including the ureter), the mesenchyme probably restricts lateral branching and provides guidance cues in vivo for directional branching and elongation as well as functioning to modulate tubular caliber and induce differentiation. Selective cadherin, claudin, and microvillar protein expression as the UB matures likely enables the formation of a tight, polarized differentiated epithelium. Although, in vivo, metanephric mesenchyme development occurs simultaneously with UB branching, these studies shed light on how (mesenchymally derived) soluble factors alone regulate spatial and temporal expression of morphogenetic molecules and processes (proliferation, apoptosis, etc.) postulated to be essential to the UB branching program as it forms an arborized structure with a continuous lumen.     

Histological markers of solid cell nests of the thyroid. With some emphasis on their expression in thyroid ultimobranchial-related tumors

It was recently demonstrated that C cells, mucosubstances, and the intermediate filament epidermal keratin are present in solid cell nests (SCN) of the thyroid. The reliability and usefulness of these markers in detecting these ultimobranchial nests have, so far, not been comparatively investigated. In the light of this, a study for the presence of these tracers in SCN at different stages of postnatal life was undertaken. The existence of carcinoembryonic antigen (CEA) in this gut-derived tissue was also searched for. Epidermal keratin was present in the epidermoid-like cells from all SCN studied; calcitonin-immunoreactive C cells and mucosubstances did not always occur. These findings reveal that this cytokeratin would remain as a potential marker to detect and trace back the ultimobranchial tissue component of the thyroid in earlier stages of development. The presence of CEA in practically all SCN surveyed would further support the view that they originate in the gut. This antigen would also be of great value for histological identification of these nests. The usefulness of these markers for the interpretation of the histogenesis of some ultimobranchial-related thyroid neoplastic growths, specifically the medullary and mucoepidermoid carcinomas, is briefly discussed.     

Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system

Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.     

TGF-beta superfamily members modulate growth, branching, shaping, and patterning of the ureteric bud

Protein-rich fractions inhibitory for isolated ureteric bud (UB) growth were separated from a conditioned medium secreted by cells derived from the metanephric mesenchyme (MM). Elution profiles and immunoblotting indicated the presence of members of the transforming growth factor-beta (TGF-beta) superfamily. Treatment of cultured whole embryonic kidney with BMP2, BMP4, activin, or TGF-beta1 leads to statistically significant differences in the overall size of the kidney, the number of UB branches, the length and angle of the branches, as well as in the thickness of the UB stalks. Thus, the pattern of the ureteric tree is altered. LIF, however, appeared to have only minimal effect on growth and development of the whole embryonic kidney in organ culture. The factors all directly inhibited, in a concentration-dependent fashion, the growth and branching of the isolated UB, albeit to different extents. Antagonists of some of these factors reduced their inhibitory effect. Detailed examination of TGF-beta1-treated UBs revealed only a slight increase in the amount of apoptosis in tips by TUNEL staining, but diminished proliferation throughout by Ki67 staining. These data suggest an important direct modulatory role for BMP2, BMP4, LIF, TGF-beta1, and activin (as well as their antagonists) on growth and branching of the UB, possibly in shaping the growing UB by playing a role in determining the number of branches, as well as where and how the branches occur. In support of this notion, UBs cultured in the presence of fibroblast growth factor 7 (FGF7), which induces the formation of globular structures with little distinction between the stalk and ampullae [Mech. Dev. 109 (2001) 123], and TGF-beta superfamily members lead to the formation of UBs with clear stalks and ampullae. This indicates that positive (i.e., growth and branch promoting) and negative (i.e., growth and branch inhibiting) modulators of UB morphogenesis can cooperate in the formation of slender arborized UB structures similar to those observed in the intact developing kidney or in whole embryonic kidney organ culture. Finally, purification data also indicate the presence of an as yet unidentified soluble non-heparin-binding activity modulating UB growth and branching. The data suggest how contributions of positive and negative growth factors can together (perhaps as local bipolar morphogenetic gradients existing within the mesenchyme) modulate the vectoral arborization pattern of the UB and shape branches as they develop, thereby regulating both nephron number and tubule/duct caliber. We suggest that TGF-beta-like molecules and other non-heparin-binding inhibitory factors can, in the appropriate matrix context, facilitate "braking" of the branching program as the UB shifts from a rapid branching stage (governed by a feed-forward mechanism) to a stage where branching slows down (negative feedback) and eventually stops.     

Identification of a novel human voltage-gated sodium channel alpha subunit gene, SCN12A

We have cloned a cDNA encoding a novel human voltage-gated sodium channel alpha subunit gene, SCN12A, from human brain. Two alternative splicing variants for SCN12A have been identified. The longest open reading frame of SCN12A encodes 1791 amino acid residues. The deduced amino acid sequence of SCN12A shows 37-73% similarity with various other mammalian sodium channels. The presence of a serine residue (S360) in the SS2 segment of domain I suggests that SCN12A is resistant to tetrodotoxin (TTX), as in the cases of rat Scn10a (rPN3/SNS) and rat Scn11a (NaN/SNS2). SCN12A is expressed predominantly in olfactory bulb, hippocampus, cerebellar cortex, spinal cord, spleen, small intestine, and placenta. Although expression level could not be determined, SCN12A is also expressed in dorsal root ganglia (DRG). Both neurons and glial cells express SCN12A. SCN12A maps to human chromosome 3p23-p21.3. These results suggest that SCN12A is a tetrodotoxin-resistant (TTX-R) sodium channel expressed in the central nervous system and nonneural tissues.     

Developmental and functional aspects of grafting of the suprachiasmatic nucleus in the Brattleboro and the arrhythmic rat

The development of suprachiasmatic nuclei (SCN) dissected from fetal rats and grafted in adult rat brains has provided additional insights in the normal ontogeny of the SCN. The SCN survives rather easily and develops to its typical adult cytoarchitectonical arrangement of contiguous clusters of vasopressin (VP)-, vasoactive intestinal polypeptide (VIP)- and somatostatin (SOM)- immunoreactive cells. Neither site of implantation, nor the establishment of efferent or afferent connections of the grafted SCN seems to be essential to allow it to develop normally into this distinguishing cytology. This independent maturation does certainly not contradict with its known endogenous and independent potency of circadian pacemaker function in the brain. If the fetal SCN is grafted in such a way that it could merge with the parenchyma of the brain of a VP-deficient Brattleboro rat, the VP neurons of the SCN often establish efferent connections with the genuine target areas of this nucleus as could be shown immunocytochemically. When the fetal SCN is grafted homotopically in the brain of SCN-lesioned rat (or hamster), the surviving SCN neurons are able to reverse the arrhythmicity of these rats. Free-running circadian rhythm of drinking or motor behaviour in constant darkness are induced within weeks after grafting. A correlation between this restorative effect and the immunocytochemical staining pattern of the SCN in the transplant and/or the afferent and efferent connections between graft and host brain, could, however, not be shown conclusively. Transplants with surviving SCN are also seen when arrhythmicity was still present, which made us conclude that there has to be a neural connection between graft and host rather than a neurohumoral control in order to explain the restorative effect of the SCN graft in SCN-lesioned animals.     

Immunoreactive substance P is not part of the retinohypothalamic tract in the rat

The retinohypothalamic tract (RHT) is a monosynaptic retinofugal pathway mediating information concerning the light/dark cycle from the retina to the brain's biological clock located in the suprachiasmatic nucleus (SCN). Light information, which daily adjusts (entrains) the rhythms of behaviour and physiology generated by the SCN, is mediated by two neurotransmitters, viz. glutamate and pituitary adenylate cyclase activating polypeptide (PACAP), co-stored in the RHT. Substance P (SP) modulates photic- and glutamate-induced phase shifts but data on its possible presence in the RHT are conflicting. By labelling the RHT projection in the SCN with the anterograde tracer cholera toxin subunit B (ChB) and antibodies against PACAP, we have shown that SP immunoreaction is absent from the PACAP/ChB-labelled nerve fibres in the SCN, indicating that the SP-immunoreactive nerve fibres are not part of the RHT but may originate from SP-immunoreactive cell bodies located within the SCN. In the retina, SP immunoreactivity occurs in amacrine cells in the inner nuclear cell layer, in a few displaced amacrine cells in the ganglion cell layer and in a dense plexus of SP-immunoreactive nerve terminals of the inner plexiform layer. Double immunostaining has revealed that SP-immunoreactive cells and fibres in the retina are not identical with the PACAP-immunoreactive ganglion cells that constitute the RHT. These findings together with the demonstration that bilateral eye enucleation does not decrease the number of SP-immunoreactive nerve fibres in the SCN indicate that SP is not a neurotransmitter in the RHT but could be an intrinsic neurotransmitter of the SCN modulating photic input to the clock.     

Ultimobranchial gland ultrastructure of larval axolotls,Ambystoma mexicanum shaw,with some observations on the newt,Pleurodeles waltlii Micahelles

Summary The fine structure of the ultimobranchial (UB) glands of two common laboratory urodeles, viz., larval axolotls, Ambystoma mexicanum Shaw and adult Pleurodeles waltlii Micahelles, is described and compared in what is believed to be the first ultrastructural report on urodele UB glands. The axolotl UB gland shows a wide variety of form, being represented by an elongated diffuse series of follicles and sometimes by one or two large discrete terminal follicular bodies. In these axolotl UB glands up to four cell categories are distinguishable including a tonofilamentous cell and a secretory cell that is possibly homologous with calcitonin-producing C cells of anurans or other vertebrates. These two cell categories are also found in the Pleurodeles gland. The possible significance of the various cells is considered.We are indebted to the Central Research Fund of London University and the Science Research Council for awards making this research possible. We would also like to thank Dr. Eyal of the Dept. of Zoology, The Hebrew University, Jerusalem for the gift of the Pleurodeles and Mr. Raynor L. Jones for his excellent technical assistance.     

Characterization of a human follicular thyroid carcinoma cell line (UCLA RO 82 W-1)

A thyroid tumor cell line has been established from the metastases of a follicular carcinoma in a female patient. Although the primary tumor released thyroglobulin (Tg) into the circulation (greater than 10,000 ng/ml), the uptake of I131 was less than 2%. After 37 replications the doubling time was 4 days and confluency was reached after 7 days from inoculation of 3 x 10(7) cells. This human thyroid tumor cell line has now been growing in culture for several years. An aneuploid chromosomal pattern was observed (62-82 chromosomes). A pair of X chromosomes was present but no Y chromosome was found which is compatible with the female origin of the cell line. EM studies revealed the presence of microvilli. Immunoperoxidase staining using specific anti-human Tg antisera indicated the presence of Tg within the cells. Nude mice developed solid-cystic tumors within 6 months after injection of the cells. The basal release of immunodetectable Tg, as measured in a perifusion system, increased in response to thyroid stimulating hormone (TSH) (P less than 0.025) or TSH combined with theophylline (P less than 0.001). Unusual isoenzyme patterns for galactose-1-phosphate-uridyltransferase (GALT) and phosphoglucomutase1 (PGM1) were detected in the tumor, compared with normal human fibroblasts and blood cells and isoenzyme patterns from the patient's lymphocytes. Because this malignant human thyroid follicular cell line has retained the ability to synthesize Tg it represents a valuable model for the study of human follicular carcinomas.     

Patterns of wheel running are related to Fos expression in neuropeptide-Y-containing neurons in the intergeniculate leaflet of Arvicanthis niloticus

A variety of nonphotic influences on circadian rhythms have been documented in mammals. In hamsters, one such influence, running in a novel wheel, is mediated in part by the pathway extending from neuropeptide-Y (NPY)-containing cells within the intergeniculate leaflet (IGL) of the thalamus to the hypothalamic suprachiasmatic nucleus (SCN). Arvicanthis niloticus is a species in which all individuals are diurnal with respect to general activity and body temperature when they are housed without a running wheel, but access to a running wheel induces a subset of individuals to become nocturnal. In the first study, the authors evaluated the possibility that nocturnal and diurnal patterns of wheel running in Arvicanthis are correlated with differences in IGL function. Adult male Arvicanthis housed in a 12:12 light-dark (LD) cycle were monitored in wheels, classified as nocturnal or diurnal, and then perfused either 4 h after lights-on or 4 h after lights-off. Sections through the intergeniculate leaflet were processed for immunohistochemical labeling of Fos and NPY. The percentage of NPY cells that expressed Fos was significantly influenced by an interaction between time of day and phenotype such that it rose from night to day in diurnal animals, and from day to night in nocturnal animals. In the second experiment, the authors established that running in a wheel actually induces Fos in the IGL of Arvicanthis. Specifically, the proportion of NPY cells expressing Fos was increased by access to wheels in nocturnal animals at night and in diurnal animals during the day. In the third experiment, the authors established that lesions of the IGL eliminate NPY fibers within the SCN, suggesting that these IGL cells project to the SCN in this species as has been established in other rodents. Together, these data demonstrate a clear difference in NPY cell function in nocturnal and diurnal Arvicanthis that appears to be caused, at least in part, by the differences in their wheel-running patterns, and that NPY cells within the IGL project to the SCN in Arvicanthis.