Ultrastructure of initial nasal process cell fusion in spontaneous and 6-aminonicotinamide-induced mouse embryo cleft lip

A search was made for cell ultrastructure differences in the initial fusion process of the medial and lateral nasal processes in mouse embryos of the following types: A/J with 12% cleft lip (CL), CL/Fr with 23% CL--both cleft-lip-predisposed strains, CL/Fr 6-aminonicotinamide (6AN)-treated (94% CL) and controls from the C57BL/6 strain (0% CL) and dancer stock (0% CL). No detectable differences were found between the A/J and CL/Fr strains and the controls in the epithelial cells showing initial contact and fusion. Epithelial surfaces not in contact in controls and where clefts were developing were smooth. Cells approaching or in contact had cell projections, intercellular junctions, desmosomes, and microfilaments demonstrating firm contact between the apposed epithelia. It has been postulated that spontaneous cleft lip was due to a predisposing face shape bringing about a failure of contact in some embryos and in others where contact was achieved fusion was normal. These data support this view. The situation, however, in 6AN-treated embryos is different. A few 6AN-treated embryos showed abnormal contact that appeared malpositioned and tenuous. The teratogen also caused increased cell death and a denser epithelium and mesenchyme. Thus 6AN-induced cleft lip could be due to the epithelial cell changes and/or to the reduction in size of the nasal processes.     

Signaling through Tgf-beta type I receptor Alk5 is required for upper lip fusion

Cleft lip with or without cleft palate is one of the most common congenital malformations in newborns. While numerous studies on secondary palatogenesis exist, data regarding normal upper lip formation and cleft lip is limited. We previously showed that conditional inactivation of Tgf-beta type I receptor Alk5 in the ectomesenchyme resulted in total facial clefting. While the role of Tgf-beta signaling in palatal fusion is relatively well understood, its role in upper lip fusion remains unknown. In order to investigate a role for Tgf-beta signaling in upper lip formation, we used the Nes-Cre transgenic mouse line to delete the Alk5 gene in developing facial prominences. We show that Alk5/Nes-Cre mutants display incompletely penetrant unilateral or bilateral cleft lip. Increased cell death seen in the medial nasal process and the maxillary process may explain the hypoplastic maxillary process observed in mutants. The resultant reduced contact is insufficient for normal lip fusion leading to cleft lip. These mice also display retarded development of palatal shelves and die at E15. Our findings support a role for Alk5 in normal upper lip formation not previously reported.     

Growth status of children treated for unilateral cleft lip and palate

Cephalometric distances, angles, and proportions were evaluated for 32 children 5 to 8 years of age treated for unilateral cleft lip and palate. The children were age and sex matched with untreated controls with normal skeletal relationships. The unilateral cleft lip and palate sample was treated by the same surgeon and orthodontist using the same techniques and appliances. Measures of overall facial proportions, facial convexity, and prognathism were not significantly different between the two groups. The primary group differences pertain to the posterior aspect of the maxilla, which is vertically short in the unilateral cleft lip and palate sample. Horizontally, the maxilla of the unilateral cleft lip and palate children was significantly longer, producing a steeper palatal plane. In addition, the zygoma and orbits of unilateral cleft lip and palate children were somewhat retruded; the posterior cranial base and total mandibular length also were longer in the unilateral cleft lip and palate children.     

Association of cleft lip and atrial septal defect in mice: a preliminary report.

Newborn A/J and CL/Fr mice with congenital cleft lip usually have an atrial septal defect of the secundum type, often associated with cyanosis. Their littermates without cleft lip rarely have a septal defect. The atrial septal defect results from a delay in growth of the atrial septum primum.     

Left cleft lip predominance and genetic similarities of L line and CL/Fr strain mice

Newborn litters of the L line and CL/Fr and A/JFr strains were examined, and sex, frequency and type of cleft lip (left, right or bilateral) were recorded. Embryos and fetuses from crosses between these strains and line were collected on days 13 to 16 of gestation, and frequency and type of cleft lip recorded. Overall cleft frequencies in L X CL/Fr, CL/Fr X L, and CL/Fr X A/JFr crosses (female stated first) were similar, while in A/JFr X L (10.3%) they were significantly lower than in L X A/JFr (23.3%). The data suggested that the same maternal effect genes were present in CL/Fr and the related L line and absent from A/JFr. In the L, CL/Fr, and A/JFr newborns, there was a tendency for males to have higher frequencies of cleft lip and bilateral cleft lip and the latter was significant for L. Left cleft lip frequency was significantly higher than right for L and CL/Fr newborns and in embryos of the CL/Fr X L and L X CL/Fr cross. No significant differences in laterality were found in the A/JFr strain, A/JFr X L, L X A/JFr, and CL/Fr X A/JFr crosses. It was concluded that (1) the embryonic and maternal effect genes for cleft lip are similar or identical in CL/Fr and L; and (2) using data from the literature, there are additional genetic factor(s) increasing left cleft lip occurrence acting in the embryo, which are present in CL/Fr, L, A/HeJ, A/He, and A/St and absent from A/JKt, A/J, A/JFr, and A/WySn.     

Interstitial deletion of the long arm of chromosome 11

A female patient with an interstitial deletion of the long arm of chromosome 11 is described. Her growth and psychomotor development were normal. She showed some facial dysmorphic features including cleft lip/palate, a probable cardiac defect and a triphalangeal thumb. A clinical correlation with similar cases is presented.     

Reduced epithelial surface activity is related to a higher incidence of facial clefting in A/WySn mice

The epithelial surfaces of the facial primordia were evaluated by scanning electron microscopy (SEM) during primary palatogenesis in two genetically related mouse strains, the A/J and the A/WySn strains. These two strains were selected because the reported frequency of spontaneous cleft lip with or without cleft palate [CL(P)] in the A/J strain approximates 0%, whereas the spontaneous frequency of CL(P) in the A/WySn strain is 20-30%. The embryos were examined prior to (two to six tail somites), during (seven to ten tail somites), and after (ten to 14 tail somites) primary palate fusion. During fusion, epithelial surface activity (characterized as cellular debris, dissociated cells, cellular projections, and epithelial bridging) was more pronounced in A/J embryos. A/WySn embryos with spontaneous cleft lip exhibited a marked deficiency in epithelial activity when compared to their normal littermates. No discernible differences were detected in the facial morphology, with the exception of the distal end of the medial nasal prominence, which appeared longer in the A/J strain. This study suggests that the degree of epithelial surface activity at the putative site of fusion and the relative length of the medial nasal prominence may account for the observed differences in facial clefting of the two strains. Face shape, related to prominence divergence, was similar in the two strains and could not explain the higher incidence of clefting observed in the A/WySn strain.     

Expression of Veratrum alkaloid teratogenicity in the mouse

Jervine, a steroidal alkaloid found as a minor constituent in the teratogenic range plant Veratrum californicum, has produced similar terata in sheep, rabbit, hamster, and chick, although the sensitivity to the alkaloid varies in the different species. Sprague Dawley rats and Swiss Webster mice are relatively insensitive. The aim of this study was to determine the teratogenic potential of jervine in three strains of mice and to ascertain if the response is strain dependent. One strain, Swiss N:GP(S), was retested since a Swiss Webster strain had been found previously to be jervine-resistant. In addition, we tested C57BL/6J and A/J, which are known to differ in their response to the teratogenic action of steroids and vitamin A. Mice were treated by gavage with single doses of jervine (70, 150, or 300 mg/kg body weight) on either day 8, 9, or 10 of gestation. Jervine was teratogenic to C57BL/6J and A/J mice but not to N:GP(S). The induced terata included cleft lip with or without cleft palate, isolated cleft palate, mandibular micrognathia or agnathia, and limb malformations. Fetal teratogenicity and maternal and fetal toxicity were highly correlated. The prevalence of each defect and fetal death was a function of strain, dose, and time of treatment. Maternal death was higher in C57BL/6J than in A/J mice. Although some of the terata were similar, the response pattern between strains was different from corticosteroids and vitamin A for both sensitive period and the strain dose response. An effect on differentiation of chondrocyte precursors may account for many of the defects, but an earlier lethal effect on differentiation of neural crest cells or precordal mesenchyme may also occur.     

Maternal cigarette smoking during pregnancy and the risk of having a child with cleft lip/palate

Maternal cigarette smoking during pregnancy as a risk factor for having a child with cleft lip/palate has been suggested by several epidemiologic studies. However, most of these studies contained small sample sizes, and a clear association between these two factors could not be established. The U.S. Natality database from 1996 and a case-control study design were used to investigate the association between maternal smoking during pregnancy and having a child with cleft lip/palate. The records of 3,891,494 live births from the 1996 U.S. Natality database were extracted to obtain cleft lip/palate cases and random controls. The National Center for Health Statistics collects maternal and newborn demographic and medical data from the birth certificates of all 50 states. New York (excluding New York City), California, Indiana, and South Dakota did not collect smoking data, and the data from these states were excluded from the analysis. A total of 2207 live births with cleft lip/palate cases were identified, and 4414 controls (1:2 ratio) were randomly selected (using the SAS program) from live births with no congenital defects. Odds ratios and 95 percent confidence intervals were determined from logistic regression models, adjusting for confounding variables, including maternal demographic and medical risk factors. A significant association was found between any amount of maternal cigarette use during pregnancy and having a child with cleft lip/palate [unadjusted odds ratio 1.55 (1.36, 1.76), p < 0.001]. Univariate analysis showed that maternal education level, age, race, and maternal medical conditions (diabetes and pregnancy-associated hypertension) were potential confounders. After adjusting for these confounders, the odds ratio remained significant [Mantel-Haenszel odds ratio 1.34 (1.16, 1.54), p < 0.001]. To determine the dose response of cigarette smoking during pregnancy, the cigarette consumption per day was divided into four groups: none, 1 to 10, 11 to 20, and 21 or more. A dose-response relationship was found when comparing each smoking category with the no smoking reference group: 1.50 (1.28, 1.76), 1.55 (1.23, 1.95), and 1.78 (1.22, 2.59), respectively. This means that increased cigarette smoking during pregnancy resulted in increased odds of having a child with cleft lip/palate. This is the largest study to date to test the association between maternal cigarette smoking during pregnancy and having a newborn with cleft lip/palate. The significant trend in the dose-response relationship strongly suggests the association of smoking tobacco and this common congenital deformity. These results emphasize the public health risks associated with smoking during pregnancy. To prevent this devastating craniofacial anomaly, educational initiatives should be considered that will alert expectant mothers to the association between smoking during pregnancy and the occurrence of cleft lip/palate.     

A morphometric analysis of craniofacial growth in cleft lip and noncleft mice.

Differences in face shape are considered a factor in cleft lip malformation. The purpose of this study was to analyze craniofacial growth in two strains: A/WySn with 28% cleft lip and C57BL/6J without cleft lip. Standardized photographs of 27 A/WySn and 25 C57BL/6J embryos with 34-46 somites (S) were taken in the superior, frontal, and lateral views. Landmarks were located and digitized for computerized analysis of growth change relative to somite number and at stages of face development before, during, and after primary palate closure. The results showed that both strains had similar overall growth patterns with increases in head width and face width, and decreases in nasal pit width. During early palatal closure in C57BL/6J mice, the nasal pit width was unchanged as brain width increased rapidly; and then later, the nasal pit width decreased as brain width increased slowly. However, during early closure in A/WySn mice, the nasal pit width decreased rapidly as brain width increased slowly; and then later, the nasal pit width was unchanged as brain width increased more rapidly. During early palatal closure, the narrower nasal pit width in A/WySn mice appeared to result from delayed growth of the supporting forebrain as the nasal pits become more medially positioned with normal face development. From the lateral view, the maxillary prominence depth was also smaller in the A/WySn strain during early palatal closure. This deficient forward growth of the maxillary prominences and the narrower positioning of the medial nasal prominences in A/WySn embryos appear to reduce the contact between the prominences and thus predispose this strain to cleft lip malformation.     

Results of multidisciplinary management of bilateral cleft lip and palate at the Iowa Cleft Palate Center.

Bilateral cleft of the lip and palate is by many standards the most complex and severe form of the defect. The complexity and severity of the defect require an unusual degree of cooperation among all specialists and especially between the surgeon and the orthodontist. There are no published findings that we know about in which comprehensive data from a number of disciplines are reported for the same group of bilateral cleft patients. Fifty randomly selected patients with bilateral complete clefts were examined by the Iowa team and two orthodontists from other institutions. The evaluations revealed that a large number of patients over the age of 10 have multiple residual problems requiring further treatment. Only 23 percent of the older patients studied were judged to have had treatment completed by the surgeon, speech pathologist, and orthodontist. It is very difficult to state whether the results obtained by our team can be considered satisfactory because there are no comparable studies that have attempted to evaluate the same parameters in multidisciplinary management.     

Morphological characteristics of monosomy X in spontaneous abortions

From a morphologic and cytogenetic study of 160 spontaneous abortion specimens with 45,X, as well as data from the literature, we conclude that monosomy X has characteristics which can suggest the diagnosis in prenatal life in the absence of a karyotype. Specimens in our study could be classified into four groups: (I) 49% consisted of a chorionic and amniotic sac, usually ruptured, containing a well-defined umbilical cord with a fragment of embryonic tissue at its end. (II) 25% consisted of small macerated embryos of 40-44 days developmental age, with pronounced retrognathia and lack of nasofrontal angle. Two specimens had a neural tube defect, one had cleft lip and palate, and two had an isolated cleft palate. The developmental age of the embryos was 5-6 weeks behind their gestational age. (III) 5% were very macerated second trimester fetuses with cystic hygromata, lymph-edema of the hands and feet, ascites, hydrothorax, and hypoplastic lungs. Four had coarctation of the aorta, three had single umbilical artery, and two had persistent left cardinal vein. Horseshoe kidney, ventricular septal defect, and anomalous subclavian artery were found once. (IV) The remaining 21% consisted only of fragments of ruptured sacs without an umbilical cord insertion site, pieces of fetal membranes or chorionic villi only. The spectrum of anomalies observed suggests that the pathogenetic mechanism for early death in 45,X embryos and fetuses may be related to vascular abnormalities or to abnormal fluid balance, leading to disturbed embryo-placental circulation, and excess fluid volume in the fetus. The 45,X karyotype is compatible with quite normal morphological development up to the fetal stage, and no specimens were found where development had ceased at a very early stage. The usual lethality of monosomy X may be explained by the presence of genes, essential for survival, on the pseudoautosomal segment of the X and Y. Lethality could result from recessive lethal mutations in this region, or from the necessity for two copies of this regions for normal development.     

Morphology of the orbital region in adults following the cleft lip-palate repair in childhood

Four measurements and two qualitative signs related to the orbits of 145 adult Caucasian cleft lip/palate patients operated on in childhood were compared with similar data on 100 normal Caucasian Canadians. The average interorbital distance in male patients with unilateral and bilateral cleft lip/palate was greater than in controls, while the interorbital distance in both male and female patients with isolated cleft palate was the same as that in controls. A hypertelorism increased interorbital distance of greater than 2 S.D. above the normal was recorded in 10 cleft patients out of 145, the maximum in male cleft patients being 48 mm and in female cleft patients 38 mm. Orbital eye fissure length asymmetry was seen only in the cleft study group while a dislocation of the eye fissure levels in the frontal plane was found both in patients with clefts and in controls. No direct relationship was found between the extent of the cleft and the incidence of hypertelorism, nor between the site of the cleft and eye fissure asymmetry in unilateral cleft lip/palate patients. The epicanthic fold was significantly more frequent in cleft lip/palate patients (28/145) than in controls (10/100). Anti-mongoloid eye fissure type was recorded only in patients with cleft but mongoloid eye fissure was present both in patients with clefts and controls.     

Induced triploidy in carp, Cyprinus carpio L.

A method for mass production of triploid carp was developed by the cold treatment of eggs after fertilization. Triploidy was demonstrated by cytophotometry and chromosome preparation techniques. Of the haploid embryos, 100% showed morphological abnormalities and died soon after hatching. Haploid embryos of maternal and paternal origin occurring in the course of cold treatment were distinguished by using a genetic marker. Among morphologically normal larvae, the frequency of triploidy was 100%, and the viability of the triploid embryos, larvae and the juvenile fish did not differ from the diploid control. Sex differentiation started in the triploid fish, but was greatly retarded. Gonad size and the small number of growing oocytes suggest that most of the triploid individuals will be sterile. Triploid carp grew at the same rate as diploid controls in the laboratory test.     

A long-term, controlled-outcome analysis of in utero versus neonatal cleft lip repair using an ovine model.

Successful open repair of a cleft lip in utero has the advantage of scarless wound healing in the fetus. Unfortunately, no long-term outcome studies have been performed to evaluate the efficacy of these repairs. Moreover, no study to date has compared the long-term results of an in utero cleft lip repair to a similar, control-matched, newborn cleft repair. This study was performed to evaluate the 9-month outcome of in utero cleft lip surgery compared with an identical cleft lip repair performed on infant lambs. In utero epithelialized cleft lips were created through an open hysterotomy in sixteen 65-day-old fetal lambs (term = 140 days) using methods described by Longaker et al. Eight of 16 animals underwent subsequent in utero repair of these clefts at 90 days gestational age. The repair of the remaining eight animals was delayed until 1 week postpartum. At 9 months, the animals were analyzed for changes in lip contour and for the degree of scarring by hematoxylin and eosin and Masson's trichrome collagen staining. Two animals in each group died from preterm labor. Of the animals that survived to term, all repaired lips had some degree of abnormality postoperatively. One of six lips repaired in utero dehisced before delivery. Three of six neonatal repairs dehisced in the first postoperative month. In the remaining animals with intact lip repairs, the vertical lip height on the repaired side was an average of 9 to 12 mm shorter than the normal lip in both the in utero and neonatally repaired animals. Phenotypically, the postnatally repaired animals had more lip distortion and visible notching. Histologically, the in utero repair was scarless and the neonatal repairs had scar throughout the entire vertical height of the lip with an associated loss of hair in this region. Maxillary growth was also evaluated. There was no inhibition of maxillary growth in the animals that underwent in utero cleft lip repair. However, in the neonatal repair group, significant maxillary retrusion was evident. Compared with the cleft side of the maxilla, horizontal growth was decreased by 11 percent (p = 0.01). Compared with the intrauterine repair group, there was a 17-percent decrease in horizontal maxillary width (p = 0.01). Straight-line in utero repair of a cleft lip produces a better long-term result in terms of maxillary growth than a similar repair performed postnatally in the ovine model. There was no diminution in maxillary growth in the animals treated in utero. Histologically, in utero repair of clefts was indeed scarless. However, both lip repairs produced lips that were significantly shorter than their contralateral noncleft sides. This degree of lip shortening would require a secondary lip revision, thereby defeating the purpose of performing an intrauterine repair. Comparisons now need to be made between in utero and neonatal repairs using a Millard-type rotation advancement technique before intrauterine treatment can be considered to be more beneficial than our current treatment modalities.     

Set of proteins shows abnormal posttranslational modification in embryos homozygous for dominant T-mutations

T and Tc are dominant mutations in the mouse that affect neuroaxial development when heterozygous and cause embryonic death when homozygous. Embryos were analyzed individually by two-dimensional gel electrophoresis at 9 1/2 days gestation, 1 day before homozygotes die in utero. A comparison of the protein patterns of mutant homozygotes with those of their littermates revealed a set of proteins (T-proteins) that showed isoelectric point (pl) polymorphism. All the T-proteins were more basic in mutant homozygotes. These polymorphisms could be detected, although they were less pronounced, in embryos as young as 7 1/2-day presomite stages, when it is impossible to distinguish homozygous mutants grossly. Interestingly, the same proteins show a pl shift from basic to acidic in wild-type embryos during development from 7 1/2 to 9 1/2 days. Thus, it appears that in T and Tc mutants a developmentally specific posttranslational acidic modification of these proteins is disturbed. The likely cause of the abnormality is a defect in some mechanism for phosphorylation, since the T-proteins of wild-type embryos were shifted to higher pls by phosphatase treatment. This disturbance appears to be localized to axial structures (neural tube, somites, and surrounding mesenchyme) since only these structures, and not the rest of the mutant homozygous embryos, contain abnormally basic T-proteins.     

Early palatal changes in complete and incomplete cleft lip and/or palate.

Early palatal development in various complete and incomplete forms of cleft lip and/or palate (CLP) was studied from birth to 3 months of age by means of dental casts. Palatal morphology (shape) and dimensions--based on reproducible reference points--were determined in a group of 128 CLP children and 68 normal children who served as controls. Substantial normal palatal growth during the first 3 months of life was observed. Round arch forms changed into oval arch forms. Growth mainly takes place in the sagittal direction (+4 mm) (transverse: +1 mm). Palates of CLP children differed significantly dependent on the type of cleft and whether the cleft was complete or incomplete. Cleft lip and alveolus children and bilateral cleft lip and palate children had more elongated palatal arches, whereas unilateral cleft lip and palate children and cleft palate children had wider palatal arches than the control group. Incomplete clefts differed from the control group in the same direction as their complete cleft forms, though less distinctly. Preoperative orthopedics used in CLP patients does not stimulate growth. On the contrary, it even restricts growth.     

From birth to maturity: a group of patients who have completed their protocol management. Part I. Unilateral cleft lip and palate

The optimal management of the cleft lip and palate patient from birth to completion of treatment presents a formidable challenge to the plastic surgeon and the associated health care system. The multidisciplinary team approach for the management of these patients is widely accepted. However, a paucity of literature exists discussing specific protocol management, interventions, and the long-term outcomes of patients who have completed a strict treatment protocol with a consistent multidisciplinary team. The aim of this study was to present the details of the specific management protocol at the Australian Craniofacial Unit for cleft lip and palate patients and to present a group of patients who have completed this specific protocol and discuss the details of their long-term care. During a 28-year period from 1974 to 2002, the records of 337 patients treated for unilateral cleft lip and palate were evaluated. Of these 337 patients, 22 have completed the same specific protocol management. The same surgeon (David, the senior author) has been responsible for performing all operative interventions and for overseeing the care of each of the 22 patients, ensuring that the treatment protocol has been executed appropriately and without deviation. The interventions and outcomes were analyzed on the basis of speech, hearing, nasal airway, occlusion, psychosocial adjustment, and appearance. Because of the large volume of data and potential differences in outcomes, the authors' intention is to present this as part I of a four-part series beginning with unilateral cleft lip and palate. The results of isolated cleft palate, isolated cleft lip, and bilateral cleft lip and palate will be presented as parts II, III, and IV, respectively. Speech results were assessed as normal speech, mild abnormality, or severe abnormality by objective measures, and intervention for velopharyngeal insufficiency was noted. Seventeen patients were rated as having normal speech. Four patients were rated as having mild speech abnormality, one patient was rated as having severe speech abnormality, and seven patients required surgery for velopharyngeal insufficiency. Hearing results were measured objectively, and good hearing results were obtained in 18 cases. Five patients required tympanoplasty. All patients required alveolar bone grafting. The high Le Fort I osteotomy was performed in six cases. Bimaxillary surgery was performed in one case. Of all the patients assessed from birth to maturity, 13 required between three and five surgical interventions, and nine required six operations or more. Further details and photographs of preoperative and postoperative examples are provided.     

Lip pressure changes following lip repair in infants with unilateral clefts of the lip and palate

The present study was designed to quantitatively assess lip pressure changes following cleft lip repair in infants with unilateral cleft lip, alveolus, and palate. Lip pressure measurements were taken using an electronic transducer system developed especially for this study. Lip pressure was monitored from 3 months (preoperatively) through 2 years of age in cleft and normal control children. Findings from the present study confirm the hypothesis that lip repair in infants with unilateral cleft lip and palate significantly increases lip pressure and that increased lip pressure remains significantly higher than in normal control children for the 2-year duration of the study. Thus increased lip pressure when the palate is unrepaired has to be considered as a factor modulating subsequent craniofacial growth.     

Genetics of Cortisone-Induced Cleft Palate in the Mouse—embryonic and Maternal Effects

Differences between mouse strains in frequency of embryonic, cortisone-induced cleft palate were examined. Probit analysis demonstrated a family of linear and parallel dose-response curves for different inbred and hybrid embryos. Since the differences between genotypes were not in the slopes of the response curves but rather in their location, it is proposed that the median effective dose (ED₅₀) of cortisone required to induce cleft palate (or the tolerance) provides a more appropriate definition of the response trait and its difference than a frequency statement. The tolerance of C57BL/6J is dominant to that of A/J. A maternal effect of A/J relative to C57BL/6J dams caused a two-fold reduction in the embryonic tolerance of cortisone. Cortisone-induced cleft palate and mortality were separate response traits.—In these and previous studies on cortisone- and other glucocorticoid-induced cleft palate in the mouse, the nature of the cleft-palate-response curve appeared to be the same for all glucocorticoids, and within-strain differences in tolerance could be used as measures of potency or bioassays for a particular effect of the glucocorticoids.