Importance of region-specific epithelial rearrangements in mouse rugae development

Epithelial appendages on palatal rugae develop during mouse palatogenesis through epithelial thickening and pattern formation. Recently, the patterned formation of nine rugae was observed together with the specific expression patterns of Shh in rodents. However, no crucial evidence was found for a direct association between Shh expression and the distinct structural formation of rugae. In order to reveal possible relationships, we investigated the morphological changes of rugae and expression patterns of Shh directly by in vitro organ culture at embryonic day 13 (E13) for 2 days. To compare and examine the diverse growing aspects of the palate and rugae, we carefully observed the detailed morphogenesis, with cell proliferation of the rugae occurring between E13 and E14.5. After 2 days of cultivation at E13, DiI micro-injections revealed that the middle part of the palate, adjacent to the upper molar-forming region, contributed to the formation of the subsequent structure of rugae by extensive cell rearrangement and proliferation within the epithelium in the preferred anteroposterior direction. The results also defined the intimate relationship between Shh expression and rugae formation.     

»Rugae« und Wachstumszonen bei Korallen

Rugae are more or less horizontal, fine ridges of the epitheca of corals. Therefore, they are rare in modern scleractinia who seldom have an epitheca. Following the studies ofBarnes (1972) the rugae are a sign for the daily increment of the sceleton. Besides the rugae there are also other zones of growth. They are shown by the radiography as an alternation of dark and light bands. This zones of different density are interpreted as a seasonal change of zones of different thickness (? and arrangement) of trabeculae. It seems possible that there is also a monthly rhythm of the growth the causes of which are unknown. In rugosa there are rugae in solitary corallits as well as in colonial corallums. The rugae of the cerioid specimen of “Columniphyllum” sulcatum described byQuenstedt 1879 have a width up to 0.3 mm. In spite of this unusually width it seems probable that these rugae are also the product of the daily increment. The difficulties of interpretation, identification and combination of rugae in determined time periods are great handicaps for their use as “geochronometric clocks”. But with more knowledge about the growth of recent corals in regard to the diverse factors of the environment, the rugae would be a possible important feature for palecologic evidences.     

Patterning of palatal rugae through sequential addition reveals an anterior/posterior boundary in palatal development

Background

The development of the secondary palate has been a main topic in craniofacial research, as its failure results in cleft palate, one of the most common birth defects in human. Nevertheless, palatal rugae (or rugae palatinae), which are transversal ridges developing on the secondary palate, received little attention. However, rugae could be useful as landmarks to monitor anterior/posterior (A/P) palatal growth, and they provide a simple model of mesenchymal-epithelial structures arranged in a serial pattern.

Results

We first determined in which order the nine mouse rugae appear during development. Our results revealed a reiterative process, which is coupled with A/P growth of palatal shelves, and by which rugae 3 to 7b are sequentially interposed, in the increasing distance between the second most anterior ruga, ruga 2, and the two most posterior rugae, rugae 8 and 9. We characterized the steps of ruga interposition in detail, showing that a new ruga forms from an active zone of high proliferation rate, next to the last formed ruga. Then, by analyzing the polymorphism of wild type and Eda^Ta mutant mice, we suggest that activation-inhibition mechanisms may be involved in positioning new rugae, like for other skin appendages. Finally, we show that the ruga in front of which new rugae form, i.e. ruga 8 in mouse, coincides with an A/P gene expression boundary in the palatal shelves (Shox2/Meox2-Tbx22). This coincidence is significant, since we also found it in hamster, despite differences in the adult ruga pattern of these two species.

Conclusion

We showed that palatal rugae are sequentially added to the growing palate, in an interposition process that appears to be dependent on activation-inhibition mechanisms and reveals a new developmental boundary in the growing palate. Further studies on rugae may help to shed light on both the development and evolution of structures arranged in regular patterns. Moreover, rugae will undoubtedly be powerful tools to further study the anteroposterior regionalization of the growing palate.     

Prenatal development of rugae palatinae in mice: scanning electron microscopic and histologic studies

The development and spatial arrangement of rugae palatinae was investigated using sagittal histological sections through the heads of 12- to 19-day mouse (ICR) embryos (appearance of vaginal plug = day 1) and scanning electron microscopy (SEM) visualization. Three distinct, consecutively occurring types of developing rugae were described: 1) rugal anlage (formed by thickened epithelium mostly burrowed in mesenchyme), 2) primitive ruga (transversally oriented band of thickened epithelium protruding to the oral cavity), and 3) definitive ruga (transversally oriented mesenchymal ridge protruding to the oral cavity, covered by epithelium). As the characteristic configuration and spatial patterns were found on each of days 13-19, rugae could be utilized as a natural positional marker (eg, in odontogenesis or palatogenesis studies). The foremost rugae start to differentiate between days 12 and 13. Between days 13 and 14 the number of rugae conspicuously increases in the anterior third of palatal shelf, and by palatal shelf horizontalization (day 15, a.m.) new rugae originate in the middle third. We presume that the origin of rugae is dependent upon inductive epithelial-mesenchymal interactions. The presence of developing rugae and the timing of their origin (occurrence of tissue interactions) in the middle versus the anterior third of the palatal shelf appears to be reflected in the mode of cellular arrangement, extracellular matrix (ECM) composition, and, probably, even in the elevation mechanism. Six stages of formation of the ruga were defined, and stage-dependent arrangements of epithelial and mesenchymal cells within the developing ruga were documented. A rearrangement of cells precedes the occurrence of the primitive ruga as well as its transformation to the definitive one. These events are discussed in relation to the hypothetical integrated function of cytoskeletal and ECM components. Regarding the developmental relation of rugae to maxillary dentition in the mouse, a comparison of particular stages of teeth and rugae development and analysis of their similarities and dissimilarities may extend the knowledge of general rules of morphogenesis and differentiation in oral biology.     

Heterochronic shifts explain variations in a sequentially developing repeated pattern: palatal ridges of muroid rodents

SUMMARY Metazoans are largely made of repeated parts, and metazoan evolution is marked by changes in the number of these parts, called meristic evolution. Understanding the mechanisms associated with meristic changes is thus a critical issue to evolutionary developmental biology. Palatal rugae are sensory ridges regularly arranged on the hard palate of mammals. They develop sequentially following mesio-distal growth of the palate, and activation–inhibition mechanisms very likely control spacing and timing of this sequential addition. In this study, we characterized trends in rugae number evolution among muroid rodents, showing that most species display 8±1 rugae, changes by one being very frequent in the phylogeny. We then compared development of three muroid species: mouse (nine rugae), rat (eight), and golden hamster (seven). We showed that palatal growth rate, spacing, and addition rate in mouse/rat were remarkably similar (with respect to the embryo size difference), and that increase to nine rugae in mouse is achieved by postponing the end of the addition process (hypermorphosis). Such a heterochronic shift may be typical of ±1 variations observed among muroid rodents. In contrast, decrease to seven rugae in golden hamster is attributed to early growth termination (progenesis) of the palate, which correlates with the severe shortening of gestation in this species. Our results provide an experimental support to the intuitive view that heterochronies are especially relevant to meristic evolution of traits that rely on a sequential addition process. We also interpret our results in the light of developmental constraints specifically linked to this kind of process.     

Shh signaling is essential for rugae morphogenesis in mice

Palatal ridges, or rugae palatinae, are corrugated structures observed in the hard palate region. They are found in most mammalian species, but their number and arrangement are species-specific. Nine palatal rugae are found in the mouse secondary palate. Previous studies have shown that epithelial Shh signaling in the palatal ridge plays an important role during rugae development. Moreover, Wnt family members, including LEF1, play a functional role in orofacial morphogenesis. To explore the function of Shh during rugae development, we utilized the maternal transfer of 5E1 (anti-Shh antibody) to mouse embryos. 5E1 induced abnormal rugae patterning characterized by a spotted shape of palatal ridge rather than a stripe. The expression patterns of Shh and Shh-related genes, Sostdc1, Lef1 and Ptch1, were disrupted following 5E1 injection. Moreover, rugae-specific cell proliferation and inter-rugae-specific apoptosis were affected by inhibition of Shh signaling. We hypothesize that the altered gene expression patterns and the change in molecular events caused by the inhibition of Shh signaling may have induced abnormal rugae patterning. Furthermore, we propose a reaction–diffusion model generated by Wnt, Shh and Sostdc1 signaling. In this study, we show that Sostdc1, a secreted inhibitor of the Wnt pathway, is a downstream target of Shh and hypothesize that the interaction of Wnt, Shh and Sostdc1 is a pivotal mechanism controlling the spatial patterning of palatal rugae.     

The common developmental origin and phylogenetic aspects of teeth, rugae palatinae, and fornix vestibuli oris in the mouse

Positional and temporal information is of fundamental importance in understanding the morphogenesis of dentition. In order to determine the fate of epithelial cells localized within specific epithelial thickened regions of the forming mouse maxilla, we analyzed serial histological sections in the frontal plane mouse embryos of 12-15 days' gestation. The epithelial thickening of the oral surface of the maxilla from 12-day embryos was spatially delineated and termed the odontogenic epithelial zone (OEZ). Beginning with 12-day embryos, analyses of camera lucida drawings indicated that the OEZ dissociates into anterior (diastema region) and posterior (molariform tooth organ region) epithelial aggregates that form plate-like configurations. The epithelial plates subsequently divide in a mediolateral direction into the epithelial anlagen of rugae palatinae, teeth, and fornix vestibuli oris superior. The medial and lateral parts of the m1 epithelial anlage are situated in dorsal continuation of both the dental and vestibular laminae of the diastema region. The anlage appears to be of dual origin. The fornix vestibuli oris superior develops from two parts: in the rima oris region from the lip-furrow lined with the vestibular lamina, and in the cheek region from the cheek-furrow in place of fusion of the maxillary and mandibular outgrowths. In 15-day embryos with well-formed secondary palates, the rugae occur, numbering nine on each palatal process. The m1 enamel organ cup excavation is positioned between the level of the fifth extending to the seventh rugae. It appears that the division of the maxillary outgrowth oral epithelial covering into rugae as well as into the dentition anlage is closely related. It is suggested that rugae, the vestibulum oris, and the dentition are developmentally and functionally related, and appear to have a common precursor in both ontogenesis and phylogenesis.     

Primary cilia in murine palatal rugae development

Periodic patterning of iterative structures is a fundamental process during embryonic development, since these structures are diverse across the animal kingdom. Therefore, elucidating the molecular mechanisms in the formation of these structures promotes understanding of the process of organogenesis. Periodically patterned ridges, palatal rugae (situated on the hard palate of mammals), are an excellent experimental model to clarify the molecular mechanisms involved in the formation of periodic patterning of iterative structures. Primary cilia are involved in many biological events, including the regulation of signaling pathways such as Shh and non-canonical Wnt signaling. However, the role of primary cilia in the development of palatal rugae remains unclear. We found that primary cilia were localized to the oral cavity side of the interplacode epithelium of the palatal rugae, whereas restricted localization of primary cilia could not be detected in other regions. Next, we generated mice with a placodal conditional deletion of the primary cilia protein Ift88, using ShhCre mice (Ift88 ^fl/fl;ShhCre). Highly disorganized palatal rugae were observed in Ift88 ^fl/fl;ShhCre mice. Furthermore, by comparative in situ hybridization analysis, many Shh and non-canonical Wnt signaling-related molecules showed spatiotemporal expression patterns during palatal rugae development, including restricted expression in the epithelium (placodes and interplacodes) and mesenchyme. Some of these expression were found to be altered in Ift88 ^fl/fl;ShhCre mice. Primary cilia is thus involved in development of palatal rugae.     

Merkel cells,corpuscular nerve endings and free nerve endings in the mouse palatine mucosa express three subtypes of vesicular glutamate transporters

The hard palate of rodents is a mucous membrane covered by a keratinized epithelium that typically contains Merkel cell (MC)-neurite complexes. MCs have engendered considerable research activity because of their involvement in mechanoreception and possibly also Merkel cell carcinomas. MCs derive from the neural crest, differentiate under control of peripheral nerve factors, are enriched in large dense core vesicles, and secrete neuropeptides and other neuroactive molecules. Upon stimulation, MC-neurite complexes produce slowly adapting type I responses. Here we emphasize that the murine hard palate is a highly differentiated sensory region, as shown by intravital staining with a styryl dye and immunocytochemistry with antibodies to vesicular glutamate transporters (VGLUTs). The entire palate contained densities of sensory endings and MC-neurite complexes, that nearly paralleled in abundance the vibrissal pads. MCs were differentially distributed in the murine palate; clusters of MCs were most abundant in the antemolar and intermolar rugae, while individual MCs were particularly enriched in the rugae at the mid-portion of the palate and in the postrugal field. VGLUT1, VGLUT2 and VGLUT3 were expressed in MCs throughout, although immunostained MCs were most frequently encountered in intermolar than antemolar rugae. The same transporters were also present in corpuscular endings at the summit of the rugae and in intraepithelial free nerve endings throughout the palate. VGLUTs presumably load glutamate into large dense core vesicles in MCs and into small clear vesicles in corpuscular and free nerve endings. The data suggest that glutamate release, or co-release, is likely to represent an important functional aspect of palatine Merkel cells and neighboring corpuscular and free nerve endings.     

The inductive role of Wnt-β-Catenin signaling in the formation of oral apparatus

Proper patterning and growth of oral structures including teeth, tongue, and palate rely on epithelial–mesenchymal interactions involving coordinated regulation of signal transduction. Understanding molecular mechanisms underpinning oral–facial development will provide novel insights into the etiology of common congenital defects such as cleft palate. In this study, we report that ablating Wnt signaling in the oral epithelium blocks the formation of palatal rugae, which are a set of specialized ectodermal appendages serving as Shh signaling centers during development and niches for sensory cells and possibly neural crest related stem cells in adults. Lack of rugae is also associated with retarded anteroposterior extension of the hard palate and precocious mid-line fusion. These data implicate an obligatory role for canonical Wnt signaling in rugae development. Based on this complex phenotype, we propose that the sequential addition of rugae and its morphogen Shh, is intrinsically coupled to the elongation of the hard palate, and is critical for modulating the growth orientation of palatal shelves. In addition, we observe a unique cleft palate phenotype at the anterior end of the secondary palate, which is likely caused by the severely underdeveloped primary palate in these mutants. Last but not least, we also discover that both Wnt and Shh signalings are essential for tongue development. We provide genetic evidence that disruption of either signaling pathway results in severe microglossia. Altogether, we demonstrate a dynamic role for Wnt-β-Catenin signaling in the development of the oral apparatus.     

Arteriovenous malformation of the stomach

A patient with chronic anemia is presented who radiologically showed prominent rugae of the stomach. Angiography demonstrated an arteriovenous malformation with a large feeding artery and prominent draining veins.     

Development and growth of palatal rugae in the mouse

Palatal rugae began to develop in the mouse, before the elevation of the palatal shelves, as localized regions of epithelial proliferation and thickening. Subsequently, fibroblasts and collagen fibres accumulated in the connective tissue subjacent to the thickened epithelium and later assumed a distinctive orientation, the fibres running anteroposteriorly within the core and in concentric curves across the base of each ruga. The role of collagen in rugal morphogenesis was examined after inhibiting its formation by feeding the lathyritic agent beta-aminopropionitrile to pregnant females. This substance markedly affected the eventual height of the rugae at birth, confirming the importance of collagen in rugal development.     

Disruption of pattern formation in palatal rugae in fetal mice heterozygous for First arch (Far)

The purpose of this study was to document the extent of disruption in the pattern of palatal rugae caused by the presence of one copy of the First arch mutation. The palatal ruga pattern was found to be disrupted in 86% of 15- to 17-day mouse fetuses that were heterozygous for the First arch mutation in the ICR/Bc strain, compared with 9% in ICR/Bc fetuses of normal (+/+) genotype. This new observation in First arch heterozygotes, together with the previously reported dominant effects of the First arch mutation, particularly the bifurcation of the maxillary nerve (100% in both BALB/cGaBc and ICR/Bc strains), the disruption of maxillary vibrissa pattern (80% in ICR/Bc), and the hemifacial deficiency (38% in ICR/Bc), has led us to redefine the First arch mutation as a semidominant, Far. Like the other defects caused by Far, the rugal defects are in tissue derived from the embryonic maxillary prominence. The rugal defects observed in +/Far palates were always asymmetrical and most often involved fragmentation and misalignment of two or more of rugae 4-7. The relatively large degree of variation in ruga pattern observed in fetuses of normal genotype suggests that it is a less well canalized trait than the normal pattern of maxillary vibrissae which varies only in a few very specific and minor ways. The First arch mutation, which in heterozygotes disrupts pattern formation in both palatal rugae and maxillary vibrissae, can be used to study genetic control of pattern formation in mammalian embryos.     

Menetrier's disease in a rhesus monkey (Macaca mulatta).

Gross and microscopic features closely resembling those found in Menetrier's disease in man are described in a 20-month-old rhesus monkey. The gastric lining was characterized by greatly enlarged rugae caused by mucosal hypertrophy and hyperplasia along with outfolding of the muscularis mucosa and the submucosa. The mucosa and submucosa were infiltrated with inflammatory cells, mainly lymphocytes and plasma cells.     

Scanning electron microscopy of exposed surfaces of the porcine placenta

The three-dimensional development of the separated parts of the porcine placenta from 9 Danish Landrace sows at gestational stages from 20 to 100 days was studied. After cautious separation of the allantochorion and the endometrium in a 1 mM buffered ethylenediaminetetraacetic acid solution, the separated parts were processed for scanning electron microscopy by routine methods. The macroscopic enlargement resulted from primary and secondary circular folds or plicae, which were permanent on the maternal side, whereas they were mainly non-permanent on the fetal side. The areolar placenta and interareolar placenta with formation of permanent microscopic folding on both sides were described. The observations of the separated parts yielded new information on the development of surface enlargement during gestation and revealed a hitherto unknown regular architecture of the endometrium by the formation of parallel primary ridges or rugae with secondary ridges or rugae at their sides subdividing the maternal troughs or fossae. This configuration on the maternal side explains the transformation of the regular chorionic ridges from the 63-day stage into bulbous protrusions at the 100-day stage. Based on these observations the precise terminology used above was proposed.     

Namibnema papillata gen. et sp.n. and Axonolaimus deconincki sp.n. (Nematoda, Axonolaimoidea) from marine and estuarine beaches of southern Africa

Namibrema papillata gen. et sp.n. is closely related to Nicascolaimus punctatus Ricmann, 1986. hnema papillutu gen. ct sp.n. is closcly related to Nicuwolaimus punctutur Ricmann. 1986. The presence of twelve stomatal rugae, the punctated cuticle and the shape of the pre-anal supplements indicate a relationship with species of the Chromadorina. Axonolaimus deconincki sp.n. is chjaracterized by the complex nature of the gubernaculum.     

西藏合腹茧蜂属一新种记述（膜翅目：茧蜂科：甲腹茧蜂亚科）

合腹茧蜂属Phanerotomella Szepligeti中国已知7种,本文报道1新种,西藏合腹茧蜂P.xizangensis,sp.nov.,并附中国已知种检索表,模式标本保存于浙江大学应用昆虫研究所。     

Larva of the leaf beetle Labidostomis longimana (Coleoptera, Chrysomelidae, Clytrinae)

The first- and the last-instar larvae of the leaf-beetle Labidostomis longimana are described in detail for the first time. The last instar larva is morphologically close to L. sibirica larva and differs from it in the absence of a projection in the medial emargination of the labrum, in the presence of 7 spiniform setae on the tibiotarsus ventrally, 11–13 narrow simple setae on the tibiotarsus dorsally, and fine rugae on the larval case.     

Mechanism of intraoral transport in macaques

All mammals have the same divisions of cyclic movement of tongue and hyoid during mastication: a protraction or forward phase that begins at minimum gape, and a retraction or return phase. Nonanthropoid mammals transport food from the oral cavity to the oropharynx during the return phase; food on the dorsal surface of the tongue moves distally while the tongue is retracted. Macaques, however, transport food during the protraction phase of tongue/hyoid movement. Food is squeezed posteriorly by contact between the tongue surface and the palate anterior to the food. This mechanism of transport is occasionally seen in nonanthropoid mammals when they are transporting liquids from the oral cavity to the oropharynx. It has, however, not been seen when these mammals transport solid food. One morphological basis for this difference is the reduction in height of the rugae of the palate of macaques. In most mammals these rugae are pronounced ridges that are able to hold food in place during protraction as the tongue slides forward beneath the food. Anthropoids and other mammals differ in the way they store food prior to swallowing. When macaques transport food to the oropharynx, usually they swallow in the next cycle, but always in the next 2 or 3 cycles. Most mammals transport and store food in the oropharynx for several cycles before a swallow clears that region of food. This behavior is correlated with differences in morphology of the oropharynx; anthropoids have reduced valleculae, the area in which other mammals store food prior to swallowing.