Dentition patterns in bilateral cleft lip subphenotypes: multicenter study.

OBJECTIVES: Here, we retrospectively investigated cases of bilateral oral clefts (OCs) to determine the clinical relevance of detailed distinction of incomplete cleft lip subphenotypes, based on morphological severity of the cleft, within the categories cleft lip with or without alveolus (CL ± A) and cleft lip, alveolus, and palate (CLAP). We further assessed possible associations between CL subphenotypes (complete vs different incomplete types) and different dentition patterns of the lateral incisor. MATERIALS AND METHODS: Our analysis included 151 non-syndromic Caucasian bilateral OC-patients (8-20 years old) from the Dutch Association for Cleft Palate and Craniofacial Anomalies registry. Six different deciduous and permanent lateral incisor patterns were distinguished: normal position (z/Z), supernumerary lateral incisor (n/N), presence in the anterior (x/X) or posterior (y/Y) segment of the cleft, one in each cleft segment (xy/XY), and agenesis (ab/AB). Logistic regression was performed to show the associations between the CL subphenotypes and dentition patterns of the lateral incisor. RESULTS: One hundred three had complete, while 48 had incomplete CLs. Patterns z/Z and n/N were associated with a submucous/vermillion notch, incomplete CL, and intact alveolus. Patterns x/X, y/Y, and xy/XY were most common in patients with two-thirds to subtotal CL and complete CL. The most severe pattern, ab/AB, was most commonly associated with complete CL. CONCLUSIONS: Based on the morphological severity of the CLs, it can be stated that the more severe the CL in bilateral CL ± A and CLAP, the more severe the abnormal pattern of the dentition. CLINICAL RELEVANCE: Further distinction of incomplete cleft lip subphenotypes (submucous/vermillion notch, one-third to two-thirds CL, two-thirds to subtotal CL) in bilateral CL ± A and CLAP has clinical relevance.

Dentition Patterns in Different Unilateral Cleft Lip Subphenotypes.

Oral clefts play an essential role in disturbed odontogenesis of the deciduous and permanent dentition, yet little is known about this relationship. We investigated, within the categories cleft lip with or without alveolus (CL ± A) and cleft lip, alveolus and palate (CLAP), whether different CL subphenotypes based on morphological severity of the cleft show different dentition patterns and whether a more detailed subdivision of the incomplete CL has clinical relevance. In this retrospective study, 345 children with nonsyndromic unilateral CL ± A and CLAP from the Dutch Association for Cleft Palate and Craniofacial Anomalies (NVSCA) registry were included to assess the association between the CL subphenotypes and lateral incisor patterns. Five different deciduous and permanent patterns of the lateral incisor were distinguished: located in normal position (pattern z/Z), in the anterior segment (pattern x/X) or in the posterior segment of the cleft (pattern y/Y), one in each segment of the cleft (pattern xy/XY), and agenesis of the lateral incisor (pattern ab/AB). Analyses were performed by using multinomial logistic regression models. Children born with a vermillion notch or a one-third to two-thirds CL were most likely to have a deciduous pattern x and a permanent pattern X, while children born with a two-thirds to subtotal CL were most likely to have deciduous pattern xy and a permanent pattern X compared to children with a complete CL that predominantly had deciduous pattern y and a permanent pattern AB. Based on the relationship of the CL morphology with the deciduous dentition, subdivision of the CL morphology into vermillion notch to two-thirds CL, two-thirds to subtotal CL, and complete CL appears to be an optimal subdivision. Our results indicate that a more detailed subdivision of the CL has clinical relevance and that critical factors in the pathogenesis of the CL are also critical for the odontogenesis.

Phosphatidylserine plasma membrane asymmetry in vivo: a pancellular phenomenon which alters during apoptosis.

The distribution of phospholipids across the two leaflets of the plasma membrane is important for many cellular processes including phagocytosis and hemostasis. In the present study we investigated the in vivo plasma membrane distribution of the aminophospholipid phosphatidylserine in mouse embryos with a novel technique employing Annexin V, a Ca2+ dependent phosphatidylserine binding protein, conjugated to fluorescein isothiocyanate and biotin. Annexin V directly applied to cryostat sections labeled the plasma membrane of all cells at the interface. In contrast, Annexin V injected intracardially into viable mouse embryos labeled almost exclusively apoptotic cells. These apoptotic cells were visible in all tissues and derived from all germ layers. Our experiments demonstrate that phosphatidylserine is asymmetrically distributed between the two leaflets of the plasma membrane in virtually all cell types in vivo and that this asymmetry is lost early during apoptosis.

The genetic background of craniosynostosis syndromes.

Craniosynostosis syndromes are developmental disorders that cause an abnormal shape of the skull due to the premature fusion of cranial sutures. Enormous progress has been made recently in understanding the genetic background of these disorders and a classification of syndromes on a genetic basis is beginning to emerge. Members of at least three gene families that play an important role in vertebrate development are associated with different craniosynostosis syndromes. Here we review the genetic aspects of this fast-moving field.

First-trimester diagnosis of Blomstrand lethal osteochondrodysplasia.

Blomstrand chondrodysplasia is a rare lethal skeletal dysplasia with presumed autosomal-recessive inheritance. A family with 2 affected fetuses was studied. One fetus demonstrated a severe skeletal dysplasia at routine transabdominal ultrasound examination at 18.5 weeks of gestation. The pregnancy was terminated and the diagnosis of Blomstrand chondrodysplasia was made at autopsy. A second affected fetus was identified by first-trimester transvaginal ultrasound at 12 weeks of gestation. In this case the diagnosis was confirmed by posttermination radiography and histopathology. From these observations, Blomstrand chondrodysplasia seems like a lethal rhizo/mesomelic short-limb, early-onset dysplasia with autosomal-recessive inheritance. Easy detectability by transvaginal ultrasound is demonstrated, but general applicability awaits further studies on the intra- and interfamilial variability of this disorder.

Case of lethal multiple pterygium syndrome with special reference to the origin of pterygia.

A case of lethal multiple pterygium syndrome is presented. Besides the anomalies usually associated with this syndrome, cardiac hypertrophy and connective tissue abnormalities were observed. On the basis of these observations, we suggest that an abnormally fragile collagen constitution is the principal disorder in the lethal multiple pterygium syndrome, which is responsible for the pathogenesis of fetal immobility, pterygia, and many of the associated anomalies.

Limb body wall malformation complex: an embryologic etiology?

Four human fetuses with the limb body wall malformation complex, also known as the amniotic band syndrome, were examined. Besides malformations of the body wall and the limb(s), anomalies of the internal organs were found in three fetuses, suggesting a disturbance of normal morphogenesis before the fifth week of development. When analyzing the observed data in relation to their etiology, no support was found for either the amniotic band or the vascular disruption theory. New insights into morphogenesis suggest that the limb body wall malformation complex results from a malfunction in the ectodermal placodes and that it can be considered to be an embryonic dysplasia.

Intracranial teratoma with multiple fetuses: pre- and post-natal appearance.

An intracranial teratoma in which six distinct dysmorphic fetuses were included was studied at autopsy. Karyotopic studies showed a normal chromosomal number in the child and the same karyotype in the three tumoral parts were examined. This is the second tumor of this type reported.

Abdominal wall defect associated with persistent cloaca. The embryologic clues in autopsy.

Three stillborn fetuses are reported in which an abdominal wall defect was associated with defects in the urogenital and anal region. Autopsy of these fetuses provided clues indicative of how and where the embryonic development went wrong. The malformation involved a disturbance of the cell deposition process, occurring in the caudal part of the embryo. During the cell deposition process, which takes place in the neural crest and the body wall placode, ectodermal cells are added to the mesodermal compartment of the embryo, thus contributing to the anlagen of several structures, including the ventral body wall. In addition, a change in the shape of the embryo is generated. The sequence of events resulting from a disturbance of the cell deposition process is explained.

mRNA and protein localization of the IGF system during mouse embryonic development in areas with apoptosis.

We analysed mRNA and protein localization of the IGF system components in regions with apoptosis during mouse development between 9.5 and 13.5 days post coitum. A spatio-temporal relationship between these expression patterns and the onset of apoptosis in specific areas was sought. The IGFBP mRNA and protein expression patterns were tissue-specific. In most tissues, mRNA expression patterns colocalized with protein localization. Discrepancies between mRNA and protein detection were found in, for example, lens, neural layer of the retina, whiskers and somites. Localization of the IGFs, the type I IGF receptor and IGFBP-2 correlated well with cell death regions. When these genes were expressed no apoptosis occurred and vice versa. Correlation of IGFBP-3, -4 and -5 with apoptosis regions was noticed only at 13.5 days post coitum. In eye muscles, whiskers and somites, the expression of IGF system components preceded the occurrence of apoptotic cells. When IGF-I expression ceased, apoptosis occurred in these areas. In conclusion, our results suggest that IGF-I, the type I IGF receptor and IGFBP-2 inhibit apoptosis. In contrast, IGFBP-3, -4 and -5 may stimulate apoptosis by trapping the IGFs. Tissue-specific modulation of IGF protective action against apoptosis by the different IGFBPs during mouse embryonal development may contribute to organ specific morphology.

MR of the caudal regression syndrome: embryologic implications.

PURPOSE: To evaluate the spectrum of developmental anomalies observed in patients with the caudal regression syndrome and relate them to the pathogenesis of this syndrome. METHODS: Nineteen children with caudal regression were investigated with MR. RESULTS: The level of vertebral agenesis varied from T-11 to S-5. In 9 of the 19 children the characteristic high-ending wedge-shaped cord terminus was observed. A separation of the anterior and posterior spinal roots of the cauda equina was observed in 9 patients. Four patients had a tethered spinal cord, in 1 in combination with a wedge-shaped cord terminus. CONCLUSIONS: The pathogenesis of the caudal regression syndrome can be divided into two kinds: there is usually a disturbance of the primary neurulation process; in other cases there is a derailment of the process of degeneration and differentiation of an initially normally developed primary and secondary neural tube. MR aids understanding of the morphology and pathogenesis of congenital malformations involved (including the associated anomalies of the genitourinary and gastrointestinal systems), but other studies are still necessary to determine the exact mechanism of this syndrome.

Embryonic development of the ventral body wall and its congenital malformations.

Congenital abdominal wall defects, frequently associated with other anomalies, are found in many forms. Consequently, there is still controversy in the literature concerning nomenclature, classification, and pathogenesis. Recently, we proposed a new nomenclature and classification of abdominal wall defects based on the early development of the umbilical cord and of the ventral body wall. According to this classification the complete spectrum of abdominal wall defects, including cloacal exstrophy, bladder exstrophy, and epispadias, can be subdivided into four types: primary (thoraco-)abdominoschisis, omphalocele, body wall dysplasia, and secondary (thoraco-)abdominoschisis. Each type is characterized by its specific configuration of the placenta, the membranes, the umbilical cord, and the fetus. Anomalies such as urachal remnants and omphalomesenteric duct malformations can be explained by disturbances during later stages of umbilical cord development.

Separation of neural and surface ectoderm after closure of the rostral neuropore.

Separation of neural and surface ectoderm after closure of the rostral neuropore in the head region has been described by investigating the integrity of the basement membranes of these epithelia in 11- to 27-somite rat embryos. The basement membranes were visualized with polyclonal antibodies against laminin. Furthermore, cell degeneration has been investigated in relation to neural crest activity, and discontinuities of the basement membrane in 9- to 30-somite mouse embryos. The separation of the basement membranes of neural and surface ectoderm in the midline is a final phase during the fusion of the neural folds, which takes place from the closure of the rostral neuropore, at the 19-somite stage, until the 27-somite stage (rat embryos), and which occurs focally with variation in the midsagittal and the transverse planes. In the prosencephalon, neural crest activity is absent during the separation phase of both epithelia, but cell degeneration may contribute to the separation of the initially connected basement membranes. A disturbance in the separation of the neural and surface ectoderm may be the pathogenetic basis of midline skull defects, and of the fronto-ethmoidal encephalocele in particular.

Cell degeneration and mitosis in the buccopharyngeal and branchial membranes in the mouse embryo.

The frequencies of cell degeneration and mitosis were investigated in the rupturing buccopharyngeal membrane (BPM) and in the persistent first branchial membrane (BM). In the BPM, cell degeneration starts many hours before rupture is visible, but mitotic figures are absent. In the BM this situation is reversed: mitotic figures are regularly observed, but a degenerating cell only occasionally. It is concluded that the ratio between the numbers of degenerating and dividing cells regulates the fate of both the BPM and the BM.

The formation of mesoderm and mesectoderm in 5- to 41-somite rat embryos cultured in vitro, using WGA-Au as a marker.

The formation of mesectodermal cells by the neural crest in 5- to 41-somite stage embryos was investigated experimentally in rat embryos cultured in vitro, using lectin-coated colloidal gold as a probe. This method labelled all ectodermal cells, among them neural crest, surface ectodermal placodal and epiblastic (primitive streak) cells. The neural crest provides the mesodermal compartment of the entire head region with cells, including the primitive cranial ganglia and the branchial arches. In the head region migration of neural crest cells over a great distance (long-distance migration) was not observed. In the trunk region neural crest derived cells were mainly found to form the primitive spinal ganglia and the sympathetic trunk, once again without long-distance cell migration. Structures and tissues that supposedly were derived from the primitive streak were hardly labelled with colloidal gold. Surface ectodermal placodes were not only found at the expected sites (e.g. epibranchial placodes) but also in the ectoderm covering the transverse septum and lateral abdominal walls.

The formation of mesoderm and mesectoderm in presomite rat embryos cultured in vitro, using WGA-Au as a marker.

Presomite rat embryos cultured in vitro were injected with the cell marker wheat germ agglutinin-gold in order to find out whether the ectoderm already formed mesodermal cells. These labelled so-called mesectodermal cells were found in all embryos studied, ranging in age from 8.7 to 9.3 days post coitum. In embryos younger than 9.0 days, the entire head fold ectoderm produced mesectodermal cells. From 9.0 days onwards, the neural crest and surface ectoderm placodes were recognizable as separate entities, both producing mesectodermal cells. The early onset of mesectodermal cell formation and the numerous and continuous manufacture led us to the conclusion that mesectodermal cells are deposited at their definitive location and that subsequent long-distance migration is unnecessary.

The cell surface coat in neurulating mouse and rat embryos, studied with lectins.

Carbohydrates in the surface coat of cells are thought to have a function in cell adhesion. The surface coat of cells, located in the fusion zone of the neural walls is investigated during neural tube closure in mammalian embryos. The presence of alpha-D-mannose, alpha-D-glucose and N-acetyl-D-glucosamine is quantified with the help of the lectins concanavalin A and wheat germ agglutinin in absence or after enzymic treatment. A two-step incubation is used, in which the second step consists of a protein-gold conjugate. A high incidence of these sugar residues was found in the fusion zone, indicating a relation to the specific capacity of these cells in establishing cell contacts.

Detection of apoptosis in ovarian cells in vitro and in vivo using the annexin v-affinity assay.

The ability of a cell to undergo apoptosis is crucial during development, tissue homeostasis, and in the pathogenesis and treatment of disease (1). To study apoptosis, it is important to be able to detect apoptotic cells reliably. Here we describe a method to detect apoptosis in vitro and in vivo on basis of the changes in phospholipid distribution in the plasma membrane that occur during this process. In healthy cells, phosphatidylserine (PS) is maintained predominantly in the inner plasma membrane surface by an aminophospholipid translocase (2). However, early during apoptosis, PS is translocated from the inner to the outer membrane surface and serves as a trigger for adjacent phagocytes to remove the dying cell (3-5). Exposure of PS can be detected in vitro and in vivo with fluorochrome- or biotin-labeled annexin V, a protein that binds to negatively charged phospholipids in the presence of calcium ions (6,7). In cells that are cultured in suspension, detection of apoptosis on the basis of PS exposure is relatively easy (8). However, sample handling of adherent cell lines, such as the ovarian cell line PA-1, might interfere with reliable detection of PS exposure. Therefore, we developed a method to detect PS exposure in adherent cell lines by labeling cells in a monolayer with annexin V and harvesting the cells afterwards by mechanical scraping (9) (Figs. 1 and 2). Fixation of annexin V-labeled cells also allows the study of the relationship between PS exposure and expression of intracellular antigens (10). We also present a method to detect apoptosis in vivo during follicular maturation in the mouse Fig. 3). This method is based on in vivo studies of viable mouse embryos, which indicate that PS exposure is a pancellular phenomenon of apoptosis during mammalian development (11,12). Fig. 1. Confocal scanning laser microscopy analysis of PA-1 ovary teratocarcinoma cells. Apoptosis was induced by treating the cells for 6 h with 50 µM roscovitine, a cyclin-dependent kinase inhibitor. Cells were labeled with annexin V-Oregon green to detect PS exposure, harvested by mechanical scraping, and labeled with propidium iodide (PI) to detect plasma membrane integrity. A and B show a linear projection of a stack of confocal images of early apoptotic cells after labeling with annexin V. At this stage, the plasma membrane integrity is preserved and, therefore, PI cannot enter the cell. C shows a secondary necrotic PA-1 cell, with clear annexin V staining at the plasma membrane (C1) and with PI staining of the condensed and fragmented chromatin (C2). Fig. 2. Dotplot of bivariate PI/annexin V flow cytometric analysis of adherent ovary cell line PA-1. Plasma membrane integrity is shown on the X-axis and annexin V immunofluorescence is shown on the Y-axis. Cells were treated with 50 µM roscovitine to induce apoptosis. 6 h after roscovitine treatment, cells were labeled with annexin V-Oregon green, harvested by scraping, and labeled with PI. Four populations of cells can be identified: region R1: vital cells (annexin V negative/PI negative), region R2: apoptotic cells (annexin V positive/PI negative), region R3: dead cells (annexin V positive/ PI positive); and region R4: damaged cells (annexin V negative/PI positive). For technical details, see ref. 9. Fig. 3. Micrographs of paraffin sections through mice ovaries that were perfused with biotinylated annexin V (A-F) or HEPES-buffer only (G and H). In A, annexin V labeled early apoptotic cells (arrowhead) and late apoptotic-pyknotic (arrow) granulosa cells are shown. During follicle maturation, initially apoptosis is absent (B). At later phases, annexin V labeled apoptotic granulosa cells (C, arrow) were observed in the primary (C) and secondary (D) follicles. Unstained pyknotic cells were also observed (C, arrowhead), presumably these cells were already located in the phagosomes before perfusion with annexin V. Also in the Graafian follicle, apoptotic cells were present in large numbers (E). F shows an enlargement of the boxed area in E. Labeled apoptotic and postapoptotic necrotic cells that have been shed into the antrum are clearly visible (asterisk), as well as unlabeled late postapoptotic necrotic cells. Labeling of ingested (arrowhead) and noningested (arrow) apoptotic cells was absent in ovaries of specimen that were perfused with HEPES-buffer only (G: overview, H: detail of boxed area in G). Scale bars equal 10 µm (A), 25 µm (C, F, and H), 50 µm (B and D) and 100 µm (E and G).

Clubbed fingers: the claws we lost?

Clubbed digits resemble the human embryonic fingers and toes, which look like the digits of a claw. Clubbed digits, thus, may represent the return of the embryonic claw and may even represent the claws man has lost during evolution, if ontogenesis really recapitulates phylogenesis. We put forward the hypothesis that secondary clubbing, like gynecomastia, is caused by a pathologic condition, which alters hormone levels in the blood, leading to the activation of 'dormant' genes, resulting in the development of an organ. However, the nature of the diseases that cause clubbing suggests that these hormones may actually be cytokines, acting as hormones. The nature of these cytokines is not known. They may be identified by comparing their blood levels or the combination of their blood levels to the presence or absence of clubbing, but also to the degree of clubbing and its disappearance after treatment of the primary disease.

Median cleft of the lower lip and mandible: case reports, a new embryologic hypothesis, and subdivision.

Median clefts of the lower lip and mandible are rare. In the literature so far, about 62 cases have been described. In addition, three more patients are presented here. These cases show a broad variation in the severity of this deformity, ranging from a simple notch in the vermillion to a complete cleft of the lip involving the tongue, the chin, the mandible, the supporting structures of the median of the neck, and the manubrium sterni. Several hypotheses concerning the pathogenesis of median clefts of the lip and mandible have been proposed. Most authors consider it to be a failure of fusion of the first pair of branchial arches or failure of mesodermal penetration into the midline. From our embryologic point of view, however, instead of paired branchial arches, only one first branchial arch develops during the early embryonic period (< or = 17 mm crown-rump length). Within this first branchial arch, two mandibular processes grow out, separated by a groove in the median. These mandibular processes do not fuse but merge during the late embryonic period (> or = 17 mm to < or = 60 mm crown-rump length). In the same developmental period, there is formation of the lip and the alveolar process and the anlage and outgrowth of one membrane bone center in each mandibular process, resulting in the formation of the mandible with its symphysis. As a consequence of the preceding, we propose the following subdivision of the median clefts of the lip and/or mandible: Hypoplasia of the mandibular processes during the early embryonic period will lead to the severest cleft of the mandible extending into the neck. During the late embryonic period, the less severe median clefts will develop. Disturbances of the outgrowth of bone centers of the mandible, resulting in nonformation of its symphysis, cause clefting of the mandible with involvement of all related soft tissues. Defects in the merging process produce just a notch of the vermilion or a higher cleft of the lower lip with or without involvement of the alveolar process of the mandible. In conclusion, the variety of the clefts in the median of the lower lip and/or mandible as well as the low rate of incidence can be explained by the embryologic hypothesis proposed here.