Prognostic significance of sarcopenia in patients undergoing esophagectomy for superficial esophageal squamous cell carcinoma.

Nononcological prognostic factors in superficial esophageal squamous cell carcinoma (SESCC) patients remain unclear. The aim of this study is to evaluate the relationship between sarcopenia and surgical outcome in patients with SESCC who had undergone definitive surgery. A total of 194 SESCC patients who had undergone thoracic esophagectomy with three-field lymphadenectomy without neoadjuvant therapy at Tokai University Hospital between January 2006 and December 2015 were analyzed retrospectively. Manual tracing using CT imaging was used to measure the cross-sectional areas of the skeletal muscle mass. The cutoff values for the skeletal muscle index used to define sarcopenia were based on the results of a previous study. Twenty-eight patients (14.4%) had sarcopenia, while the remaining 166 patients (85.6%) did not. A multivariate analysis suggested that sarcopenia was an independent risk factor for postoperative pulmonary complications (OR = 3.232, P = 0.026). The overall survival rate and the disease-free survival rate were both significantly worse in the sarcopenia group than in the nonsarcopenia group (P < 0.001). In a multivariate analysis, sarcopenia was an independent prognostic factor affecting overall survival (HR = 7.121, P < 0.001) and disease-free survival (HR = 6.000, P < 0.001). Patients with sarcopenia and lymph node metastasis (n = 18) had a worse outcome than the other patients (P < 0.001). This study suggests that the alleviation of sarcopenia through nutritional support and rehabilitation in SESCC patients scheduled to undergo surgery might help to prevent postoperative pulmonary complications and to improve the long-term outcome.

Prognostic impact of lymphovascular invasion in lymph node-negative superficial esophageal squamous cell carcinoma.

The relationship between lymphovascular invasion (LVI) and prognosis in patients with superficial esophageal squamous cell carcinoma (SESCC) is unclear. The aim of this study is to evaluate prognostic factors in patients with lymph node-negative SESCC. A total of 195 patients with pathologically confirmed T1a-MM, T1b, and lymph node-negative SESCC were retrospectively reviewed in this study. Overall, the disease-free survival (DFS) rate was poorer in the lymphatic invasion-positive group than in the lymphatic invasion-negative group (p = 0.002) and a multivariate analysis suggested that lymphatic invasion was the only independent prognostic factor of DFS in patients with lymph node-negative SESCC (HR = 4.075, p = 0.005). Distant organ recurrence occurred in one patient (1/52, 1.9%) in the T1b-SM2 group and in six patients (6/61, 9.7%) in the T1b-SM3 group; all of these patients had LVI. LVI-positive patients had a poorer DFS than invasion-negative patients in the T1b-SM2 and SM3 groups (p = 0.026), and a multivariate analysis suggested that LVI was the only independent prognostic factor of DFS in patients with lymph node-negative SM2 and SM3 SESCC (HR = 5.165, p = 0.031). Lymph node-positive patients had a significantly poorer DFS rate than lymph node negative and LVI positive patients among the SM2 and SM3 SESCC patients (p = 0.018). The present results suggested that LVI was an independent prognostic factor in patients with SM2 and SM3 lymph node-negative SESCC; however their prognosis was not worse than that of patients with lymph node-positive SM2 and SM3 SESCC, for whom adjuvant therapy is indicated as a standard treatment.

Clinicopahological features of superficial basaloid squamous cell carcinoma of the esophagus.

Basaloid squamous cell carcinoma (BSC) of the esophagus is classified as an epithelial malignant tumor and is a rare variant of squamous cell carcinoma (SCC). Most previous reports have suggested that advanced BSC has a poorer prognosis than typical SCC because of its high biological malignancy, but the biological activity of superficial BSC remains unclear. Twenty cases of superficial BSC, which underwent surgical resection in Tokai University Hospital between January 2004 and December 2013, were analyzed retrospectively. Among these cases, 19 cases with a T1 depth of invasion (BSC group) were compared with 180 cases of SCC that were resected during the same period and were pathologically diagnosed as T1 (SCC group). The frequency of lymph node metastasis in the T1 BSC group was significantly lower (2 patients, 11%) than that in the SCC group (84 patients, 47%) (P = 0.005). The frequency of lymphatic invasion in the BSC group was also lower (9 patients, 47%) than that in the SCC group (131 patients, 73%) (P = 0.021). The pathological type of the metastatic lymph node was BSC in all the superficial BSC cases with lymph node metastasis. This study demonstrated that lymph node metastasis was less likely to occur in cases with superficial BSC than in cases with superficial SCC.

Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome.

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

NaCl responsive taste cells in the mouse fungiform taste buds.

Previous studies have demonstrated that rodents' chorda tympani (CT) nerve fibers responding to NaCl can be classified according to their sensitivities to the epithelial sodium channel (ENaC) blocker amiloride into two groups: amiloride-sensitive (AS) and -insensitive (AI). The AS fibers were shown to respond specifically to NaCl, whereas AI fibers broadly respond to various electrolytes, including NaCl. These data suggest that salt taste transduction in taste cells may be composed of at least two different systems; AS and AI ones. To further address this issue, we investigated the responses to NaCl, KCl and HCl and the amiloride sensitivity of mouse fungiform papilla taste bud cells which are innervated by the CT nerve. Comparable with the CT data, the results indicated that 56 NaCl-responsive cells tested were classified into two groups; 25 cells ( approximately 44%) narrowly responded to NaCl and their NaCl response were inhibited by amiloride (AS cells), whereas the remaining 31 cells ( approximately 56%) responded not only to NaCl, but to KCl and/or HCl and showed no amiloride inhibition of NaCl responses (AI cells). Amiloride applied to the basolateral side of taste cells had no effect on NaCl responses in the AS and AI cells. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments indicated that ENaC subunit mRNA was expressed in a subset of AS cells. These findings suggest that the mouse fungiform taste bud is composed of AS and AI cells that can transmit taste information differently to their corresponding types of CT fibers, and apical ENaCs may be involved in the NaCl responses of AS cells.

Optical imaging of mouse articular cartilage using the glycosaminoglycans binding property of fluorescent-labeled octaarginine.

OBJECTIVE: The aim of the current study was to examine the cartilage-specific binding property of polyarginine peptides (R4, 8, 12, and 16) and specifically to test octaarginine peptides for the optical imaging of articular cartilage in experimentally induced arthritis in mice. METHODS: Four rhodamine-labeled polyarginine peptides each with a different-length arginine chain (R4, 8, 12, or 16) were injected into the knee joints of C57BL/6J mice (n=20). The joints were excised 1h later and the fluorescent signal intensity in cartilage cryosections was compared for the four peptides. To examine the substrate of R8 in cartilage, femoral condyles obtained from another set of mice were treated with chondroitinase ABC (Ch'ase ABC), keratanase or heparitinase then immersed in R8-rhodamine. Fluorescent signals were examined by fluorescent microscopy. Next, R8-rhodamine was injected into the right knee joints of three control and three collagen antibody-induced arthritis (CAIA) mice, and fluorescent intensity in normal and degenerative cartilage was semi-quantitatively analysed on the histological sections using image software. Finally, femoral condyles from normal mice (n=2) and CAIA mice (n=2) were immersed in R8-rhodamine and calcein, then imaged using optical projection tomography (OPT). RESULTS: Fluorescent signals were specifically detected in the cartilage pericellular matrix from the surface to the tide mark but were completely absent in the calcified layer or bone marrow. The number of arginine residues significantly influenced peptide accumulation in articular cartilage, with R8 accumulating the most. The fluorescent signal in the femoral condylar cartilage diminished when it was treated with Ch'ase ABC. R8 accumulation was significantly decreased in the degenerative cartilage of CAIA mice, and this was demonstrated both histologically and in three-dimensional (3D)-reconstruction image by OPT. CONCLUSION: R8 may be a useful new experimental probe for optical imaging of normal and arthritic articular cartilage.

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes.

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.

Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.

Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.

Decrease in L-type pyruvate kinase activity in rat liver by some promoters of hepatocarcinogenesis.

A probably promoter-specific decrease of L-type pyruvate kinase (L-PK) in Wistar rat liver is described. The possibility of utilizing the decrease in L-PK activity for screening of hepatic promoters is discussed. A significantly decreased level of activity of L-PK was observed during continuous feedings of the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene [(3'-MeDAB) CAS: 55-80-1], which initiates and promotes hepatocarcinogenesis, and of the known hepatic promoters phenobarbital [(PB) CAS: 50-06-6] and dichlorodiphenyltrichloroethane (CAS: 50-29-3) for at least 4 weeks. In contrast, if it occurred, the decrease in L-PK activity by the nonpromoting agents amobarbital (CAS: 57-43-2) and diphenylhydantoin. (CAS: 57-41-0) was temporary and almost overcome by the 4th week. The depression of L-PK activity caused by PB was reversible, was inversely correlated with PB concentration in the diet, and seemed to be organ-specific. Although hepatic promoters lowered L-PK activity in this study, data are so limited that a much more extensive study is necessary before a general conclusion can be drawn. In contrast to L-PK activity, the activity of K-type pyruvate kinase (K-PK) was induced by injections of the carcinogen diethylnitrosamine (CAS: 55-18-5) or the hepatotoxin CCl4 (CAS: 56-23-5) or by the feeding of 3'-MeDAB. However, feeding of PB or 2-methyl-4-dimethylaminoazobenzene (CAS: 54-88-6), which initiates but does not promote hepatocarcinogenesis, did not increase K-PK activity.

Evaluation of a histologic classification of mouse liver tumors based on pyruvate kinase isozymes and status of host lipids.

The histologic classification of mouse liver tumors, i.e., hepatic nodules type 1 and type 2 and hepatocellular carcinomas, was evaluated by a comparison of several biologic and biochemical markers that have been shown to be useful for the grading of tumor malignancy. The liver tumors were induced by N,N'-2,7-fluorenylenebisacetamide (CAS: 304-28-9; N,N'-fluoren-2,7-ylenebisacetamide) administration to male CD-1 mice. The ability to induce L-type pyruvate kinase activity in response to a high-carbohydrate diet disappeared in almost all the liver tumors. However, fairly good correlations were observed between the histologic classification and the relative weights of liver and intraperitoneal fat pads, between the histologic classification and the level of serum total cholesterol, and between the histologic classification and the K-type pyruvate kinase activity. The results suggest that the present histologic classification reflects the degree of tumor malignancy, and therefore, it would be useful for the classification of mouse liver tumors.

Association of minisatellite instability with c-myc amplification and K-ras mutation in methylcholanthrene-induced mouse sarcomas.

Instability of microsatellite sequences are frequently found in human tumors. In addition, minisatellite sequences, another group of highly unstable sequences, serve as sensitive markers of genetic instability. We have studied minisatellite instability in methylcholanthrene-induced mouse sarcomas. These sarcomas frequently carry the amplified c-myc gene. Seven sarcomas without the amplification and seven others with the amplification were selected randomly. Regardless of the state of the c-myc gene amplification, these sarcomas exhibited a varying degree of transplantability in syngeneic mice. The hypervariable mouse minisatellite locus Ms6hm was found to be highly unstable, specifically among sarcomas with the amplified c-myc gene. However, chromosome instability, as analyzed by micronucleus assay, was observed similarly for two groups of sarcomas. In addition, transversion of G to C and A to T was detected at the K-ras gene in four of the seven sarcomas with the amplified c-myc gene, and these mutations are thought to be induced directly by methylcholanthrene. Thus, concomitant occurrence was observed for three seemingly unrelated mutations, amplification of the c-myc locus, point mutation of the K-ras gene, and instability at the hypervariable mouse minisatellite locus. The present study indicates a possible involvement of K-ras mutation and c-myc amplification in induction of genetic instability in methylcholanthrene-induced mouse sarcomas.

Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia.

Gene targeting studies in mice have shown that the lack of Ikaros activity leads to T-cell hyperproliferation and T-cell neoplasia, establishing the Ikaros gene as a tumor suppressor gene in mice. This prompted us to investigate whether mutations in Ikaros play a role in human hematological malignancies. Reverse transcription-PCR was used to determine the relative expression levels of Ikaros isoforms in a panel of human leukemia/lymphoma cell lines and human bone marrow samples from patients with hematological malignancies. Among the cell lines examined, only BV-173, which was derived from a chronic myelogenous leukemia (CML) patient in lymphoid blast crisis, overexpressed the dominant-negative isoform, Ik-6. In 9 of 17 samples of patients in blast crisis of CML, Ikaros activity had been reduced either by drastically reducing mRNA expression (4 of 17) or by overexpressing the dominant-negative isoform Ik-6 (5 of 17). Significantly, expression of Ikaros isoforms seemed normal in chronic phase CML patients and patients with other hematological malignancies. In some cases, overexpression of the dominant-negative Ik-6 protein was confirmed by Western blot analysis, and Southern blot analysis indicated that decreases in Ikaros activity correlated with a mutation in the Ikaros locus. In summary, these findings suggest that a reduction of Ikaros activity may be an important step in the development of blast crisis in CML and provide further evidence that mutations that alter Ikaros expression may contribute to human hematological malignancies.

Fer tyrosine kinase oligomer mediates and amplifies Src-induced tumor progression.

c-Src is upregulated in various human cancers, suggesting its role in malignant progression. However, the molecular circuits of c-Src oncogenic signaling remain elusive. Here we show that Fer tyrosine kinase oligomer mediates and amplifies Src-induced tumor progression. Previously, we showed that transformation of fibroblasts is promoted by the relocation of c-Src to non-raft membranes. In this study, we identified Fer and ezrin as non-raft c-Src targets. c-Src directly activated Fer by initiating its autophosphorylation, which was further amplified by Fer oligomerization. Fer interacted with active c-Src at focal adhesion membranes and activated Fer-phosphorylated ezrin to induce cell transformation. Fer was also crucial for cell transformation induced by v-Src or epidermal growth-factor receptor activation. Furthermore, Fer activation was required for tumorigenesis and invasiveness in some cancer cells in which c-Src is upregulated. We propose that the Src-Fer axis represents a new therapeutic target for treatment of a subset of human cancers.

Active synthesis of glycosaminoglycans by liver parenchymal cell in primary culture.

Rat liver parenchymal cells were evaluated after 2 days of primary culture for their ability to synthesize and accumulate heparan sulfate as the major component and low-sulfated chondroitin sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid as the minor ones. The newly synthesized glycosaminoglycans secreted into the medium were different from those remaining with and/or the cell layer. Low-sulfated chondroitin 4-sulfate, a major glycosaminoglycan in blood, was synthesized in the order of 320 microgram/liver per day, more than 90% of which was secreted into the medium with 16 h and 40% of the glycan secreted was degraded during that time. On the other hand, heparan sulfate, the major glycosaminoglycan synthesized by the parenchymal cells, was mainly distributed in the cell layer. After 8 days of culture, the synthesis of glycosaminoglycans by the cells increased markedly, especially dermatan sulfate, chondroitin sulfate, chondroitin sulfate and hyaluronic acid.

The complete primary structure of the long form of mouse alpha 1(IX) collagen chain and its expression during limb development.

Type IX collagen is a newly discovered collagen molecule that is associated with Type II-containing collagen fibrils in cartilage, vitreous and embryonic cornea. It consists of three distinct chains: alpha 1(IX), alpha 2(IX) and alpha 3(IX). The alpha 1(IX) chain has been to be synthesized in two different forms, which are generated by alternative transcription and splicing. In this manuscript we describe the isolation and sequencing of a cDNA coding for the entire coding region of the long form of mouse alpha 1(IX) chain. Nucleotide sequence analysis of this cDNA determined for the first time the primary structure of the entire long form of the mouse alpha 1(IX) chain. RT-PCR was used to examine collagen gene expression during limb development from day 10 to 18 in mouse embryos. Collagen I and II mRNA levels gradually increased all through the developmental stages. Collagen X expression increased further after day 16 in limb development, whereas the alpha 1(IX)mRNA level dropped at this time. This could be due to active bone formation relative to cartilage synthesis in the embryonic limb bud around day 16 in mouse development.

Glycosaminoglycan synthesis by liver parenchymal cell clones in culture and its change with transformation.

Albumin-producing rat liver parenchymal cell clones (BB and BC) and their subclones in the confluent culture synthesized heparan sulfate as the major component and dermatan sulfate, chondroitin sulfate and hyaluronic acid as the minor ones. Their relative contents were similar to those present in the rat liver. Analyses of glycosaminoglycans synthesized by subclone cells (BB1S) at various cell densities, cell growth phases and passage levels have shown that relative content of heparan sulfate remained constant, suggesting that the epithelial cell possesses a stable heparan sulfate-producing capacity. On the other hand, the level of hyaluronic acid production was high at low cell density, though it remained constant during cell proliferation. Chemically transformed rat liver parenchymal cells (M) produced relatively higher amount of chondroitin sulfate than non-transformed cells did, as observed with 4-nitroquinoline-1-oxide-transformed 3T3 cells, compared to 3T3 714 cells. The results obtained on this study strongly suggest that the liver parenchymal cells synthesize a major part of the glycosaminoglycans of the liver and chondroitin sulfate production is closely related to cellular proliferations.

cDNA sequence and expression of the mouse alpha1(V) collagen gene (Col5a1).

Several overlapping cDNA clones corresponding to the entire coding sequence of the mouse alpha1(V) collagen gene (Col5a1) were isolated. The conceptual amino acid translation indicated a high degree of sequence identity (94%) with the human alpha1(V) chain. All of the important structures previously noted in the human alpha1(V) chain were also conserved in the mouse chain. The alpha1(V) transcripts were easily detected in mouse embryos as early as 11 days post coitum (d.p.c.). The transcripts were widely distributed in non-cartilaginous and cartilaginous tissues. Finally, we calculated the ratio of transcripts of alpha1(V):alpha2(V):alpha1(XI) in the calvaria and tongue of 18 d.p.c. embryos using the competitive reverse transcription-polymerase chain reaction (RT-PCR) technique. The results raised the possibility that there are at least two different kind of types V/XI collagen heterotrimers in mouse embryonic tissues.

Shh and Ptc are associated with taste bud maintenance in the adult mouse.

In mammals, taste receptor cells are organized into taste buds on tongue. Taste buds are trophically maintained by taste neurons and under continuous renewal, even in adults. We found that the receptor for Sonic hedgehog (Shh), Patched1 (Ptc), was expressed around taste buds where cells were proliferating, and that Shh was expressed within basal cells of taste buds. Denervation caused the loss of Shh and Ptc expression before the degeneration of taste buds.

Mammalian craniofacial embryology in vitro.

Our review demonstrates that the whole embryo culture system established by New and his colleagues, in combination with beneficial fluorescent dye cell-tracing techniques, has greatly contributed to many advancements in the field of mammalian craniofacial embryology, especially with regard to elucidating the developmental behavior of cephalic crest cells. In addition, based on recent results, further combining whole embryo culture with mandibular organ culture methods has allowed us to trace cranial crest cells for a much longer developmental period, i.e., presently up to the cap stage in odontogenesis.

Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation.