The Grooved Rodent Incisor Recapitulates Rudimentary Teeth Characteristics of Ancestral Mammals.

It is known from the paleontology studies of eutherian mammals that incisor numbers were reduced during evolution. The evolutionary lost incisors may remain as vestigial structures at embryonic stages. The recapitulation of the incisor patterns among mammalian species will potentially uncover the mechanisms underlying the phenotypic transition of incisors during evolution. Here, we showed that a minute tooth formed in the presumptive groove region of the gerbil upper incisor at the early developmental stages, during which multiple epithelial swellings and Shh transcription domains spatiotemporally appeared in the dental epithelium, suggests the existence of vestigial dental primordia. Interestingly, when we trimmed the surrounding mesenchyme from incisor tooth germs at or before the bud stage prior to ex vivo culture, the explants developed different incisor phenotypes ranging from triplicated incisors, duplicated incisors, to Lagomorpha-like incisors, corresponding to the incisor patterns in the eutherian mammals. These results imply that the phenotypic transition of incisors during evolution, as well as the achievement of ultimate incisors in adults, arose from differential integrations of primordia. However, when the incisor tooth germ was trimmed at the cap stage, a grooved incisor developed similar to the normal condition. Furthermore, the incisor tooth germ developed a small but smooth incisor after the additional removal of the minute tooth and a lateral rudiment. These results suggest that multiple dental primordia integrated before the cap stage, with the labial primordia contributing to the labial face of the functional incisor. The minute tooth that occupied the boundary of the 2 labial primordia might be implicated in the groove formation. This study sheds light on how rudiments incorporate into functional organs and aids the understanding of incisor evolution.

Ossification in nude mice. 1. Macroscopical study.

Ossification was studied in cleared fetuses, newborns, 1- and 6-week-old nu/nu and nu/+ mice of the B 10 LP background. The same ossification pattern was observed in nu/nu and nu/+ in the embryonal period as well as in newborn animals and mice aged 1 or 6 weeks. On the other hand, 6-week-old nu/nu mice exhibited a lower X-ray density of the skeleton and a lower thickness of the proximal tibial growth plate than 6-week-old nu/+ litter mates. The results indicate the influence of postnatal factors on the skeletal development of nu/nu mice.

Morphometric study on the fetal thyroid gland in the nude mouse (BALB/cAnNCrj-nu/nu).

Fetal thyroid glands of athymic nude mice (BALB/cAnNCrj-nu/nu) were examined morphometrically on day 18 of gestation. Compared to euthymic litter mate controls (BALB/ cAnNCrj-nu/+), both the cell height and diameter of thyroid follicles were significantly smaller; fewer well-developed follicles were found in the peripheral region of the thyroid; the body weight and total volume of the thyroid gland were also smaller in nude mice. These results suggest underdevelopment of the fetal thyroid gland of the athymic nude mouse.

Underdevelopment of fetal thyroid follicular cells in athymic nude mouse (BALB/cAnNCrj-nu/nu) observed by electron microscopic morphometry.

Fetal thyroid follicular cells of congenitally athymic nude mouse (BALB/cAnNCrj-nu/nu) were studied with an electron microscope. The area of the entire cell, nucleus and mitochondrion were measured and compared in athymic and euthymic fetal nude mice (BALB/cAnNCrj-nu/+) at 18 days of gestation. The mean area of cytoplasm was significantly smaller in homozygous athymic nude mice than in heterozygous euthymic ones. The mean area of the mitochondrion was also smaller in homozygous athymic nude mice, but the difference was not statistically significant. There was no significant difference between the two groups in the area of the nucleus. These findings suggest that the thyroid gland of athymic nude mice is still underdeveloped at the end of gestation as compared to that of their euthymic littermates.

Examination of the mouse embryo by micro-CT.

Micro X-ray computed tomography (micro-CT) is widely used in preclinical studies of small animals. However, due to the low soft tissue contrast, segmentation of soft tissues in the micro-CT image is a challenging problem. To gain a better understanding of the macroscopic anatomy of the mouse embryo, 3 fixation methods and 3 metal stainings were examined for micro-CT using C57BL/6J mouse embryos in the present study. The examination demonstrated that 1% acetic acid/95% ethanol fixative together with zinc staining provided a high contrast micro-CT image, enabling the segmentation of soft tissues. Then, using this condition, the macroscopic embryo structure of the nude mouse was examined, revealing lack of a thymus. It appears that micro-CT with the fixation and staining condition devised in the present study could be a powerful tool in detecting the effects of various mutations at embryonic stages.

An electron microscopic study of the development of the thymus anlage of athymic (nu/nu) mice.

Using electron microscopy, we examined the anlage of dysgenetic embryo thymuses of nu/nu mice aged 14.5-15 days, 16 days and 18 days p.c., with thymus anlage of nu/+ mice serving as controls. On comparing both types, we noted differences at all intervals which increased in a time-related manner. Lymphoid cells were found only very occasionally in dysgenetic thymus anlage up to day 16 p.c. while there were no capillaries at all in any of the stages investigated. In epithelial reticular cells, undemarcated (membrane non limited) spaces of varying size and containing PAS+ material were found in the cytoplasm (glycogen-like particles seen in electron microscopy). Based on our findings, and on general assumptions, we believe that the primary process occurring in the dysgenetic thymus is abnormal epithelial stroma differentiation resulting not only in a lack of lymphoid cells but, also, in a limited or probably absent vascular bed.

Molar odontogenesis in the hairless mouse.

Molar odontogenesis was studied in the hairless mouse from the initiation of the dental lamina through apposition. The dental lamina stage of the first molar was recognized on the 13th day, the bud on day 14th, cap on the 16th, bell on the 18th and apposition on the 20th day after conception. The morphology of the various stages and their temporal sequence were compared with those of other rodents.

Nude mouse embryo: ectodermal nature of the primordial thymic defect.

Sterile female homozygous nude mice were rendered fertile through a graft of thymic epithelium from heterozygous littermates 3 days after birth. Homozygous nude embryos were compared with embryos age (8-18 days). In nude mice, the embryological anomaly is noticeable from the 11th day of gestation and essentially consists of a failure of the ectoderm from the 3rd branchial cleft to proliferate and differentiate into a cervical vesicle. As a result, the endoderm from the 3rd pouch degenerates into cystic formations instead of differentiating.

The development of the thymus in the nude mouse.

The developing thymus of nude mouse embryos (derived from homozygous, nu/nu x nu/nu, matings) has been examined from 13 days' gestation onwards for the presence of large basophilic stem cells or their lymphoid progeny. No trace of either stem cells or lymphocytes has been found at any stage, indicating that from the earliest stages of thymic development lymphopoiesis is defective. However, histological evidence alone is not sufficient to rule out the possibility that small numbers of lymphocytes may be present in the nude thymus. Recent evidence that nude mice born of heterozygous (nu/+) females possess some T lymphocytes and "T lineage" cells is discussed in relation to these findings. A series of morphological changes have been defined within the epithelial component of the developing nude thymus, further analysis of which may help to determine the nature of the thymic defect.

New member of the winged-helix protein family disrupted in mouse and rat nude mutations.

Mutations at the nude locus of mice and rats disrupt normal hair growth and thymus development, causing nude mice and rats to be immune-deficient. The mouse nude locus has been localized on chromosome 11 (refs 3, 4) within a region of < 1 megabase. Here we show that one of the genes from this critical region, designated whn, encodes a new member of the winged-helix domain family of transcription factors, and that it is disrupted on mouse nu and rat rnuN alleles. Mutant transcripts do not encode the characteristic DNA-binding domain, strongly suggesting that the whn gene is the nude gene. Mutations in winged-helix domain genes cause homeotic transformations in Drosophila and distort cell-fate decisions during vulval development in Caenorhabditis elegans. The whn gene is thus the first member of this class of genes to be implicated in a specific developmental defect in vertebrates.

Association between mouse nude gene expression and the initiation of epithelial terminal differentiation.

Loss-of-function mutations in Whn (Hfh 11), a winged-helix/forkhead transcription factor, result in the nude mouse phenotype. To determine the whn expression pattern during development, we utilized mice in which a beta-galactosidase reporter gene was placed under the control of the wild-type whn promoter by homologous recombination (M. Nehls et al., 1996, Science 272, 886-889). Sites of reporter expression were confirmed by immunohistochemical staining for Whn protein or by in situ hybridization for whn mRNA. At all developmental stages, whn expression is restricted to epithelial cells. In addition to the skin and thymus, whn is expressed in the developing nails, nasal passages, tongue, palate, and teeth. In embryonic epidermis, suprabasal cells induce whn expression at the same time that terminal differentiation markers first appear. As the epidermis matures, whn promoter activity is found primarily in the first suprabasal layer, which contains keratinocytes in the early stages of terminal differentiation. In developing and mature anagen hair follicles, whn is expressed at high levels in the postmitotic precursor cells of the hair shaft and inner root sheath. Though principally associated with terminal differentiation, whn expression is also detected in progenitor cell compartments; in the hair bulb matrix and basal epidermal layer, a small subclass of cells expresses whn, while in the outer root sheath, whn promoter activity is induced as the follicle completes its elongation. Within these compartments, rare cells exhibit both whn expression and the nuclear proliferation marker Ki-67. The results suggest that whn expression encompasses the transition from a proliferative to a postmitotic state and that whn regulates the initiation of terminal differentiation.