Hindbrain neuropore tissue geometry determines asymmetric cell-mediated closure dynamics in mouse embryos.

Gap closure is a common morphogenetic process. In mammals, failure to close the embryonic hindbrain neuropore (HNP) gap causes fatal anencephaly. We observed that surface ectoderm cells surrounding the mouse HNP assemble high-tension actomyosin purse strings at their leading edge and establish the initial contacts across the embryonic midline. Fibronectin and laminin are present, and tensin 1 accumulates in focal adhesion-like puncta at this leading edge. The HNP gap closes asymmetrically, faster from its rostral than caudal end, while maintaining an elongated aspect ratio. Cell-based physical modeling identifies two closure mechanisms sufficient to account for tissue-level HNP closure dynamics: purse-string contraction and directional cell motion implemented through active crawling. Combining both closure mechanisms hastens gap closure and produces a constant rate of gap shortening. Purse-string contraction reduces, whereas crawling increases gap aspect ratio, and their combination maintains it. Closure rate asymmetry can be explained by asymmetric embryo tissue geometry, namely a narrower rostral gap apex, whereas biomechanical tension inferred from laser ablation is equivalent at the gaps' rostral and caudal closure points. At the cellular level, the physical model predicts rearrangements of cells at the HNP rostral and caudal extremes as the gap shortens. These behaviors are reproducibly live imaged in mouse embryos. Thus, mammalian embryos coordinate cellular- and tissue-level mechanics to achieve this critical gap closure event.

An Unusual First-Trimester Ultrasound Presentation of the Acrania-Anencephaly Sequence: The "Turkish Turban" Sign.

A previously unrecognized first-trimester presentation of the acrania-anencephaly sequence is described. Ultrasound features included a constriction ring around the external base of the developing skull and an enlarged globular head, resembling a Turkish turban, with large cystic spaces replacing the brain. This constellation of findings was noted in 3 first-trimester fetuses. In 2 of them, it was possible to identify the amniotic membrane attached to the constriction ring. One case presented with anencephaly and fetal demise at 16 weeks. The other 2 women terminated the pregnancies and aborted anencephalic fetuses. This subtype of the acrania-anencephaly sequence could represent an earlier segmental rupture of the amnion, which subsequently entraps the developing fetal skull.

Midline craniofacial malformations with a lipomatous cephalocele are associated with insufficient closure of the neural tube in the tuft mouse.

BACKGROUND: Genetic variations affecting neural tube closure along the head result in malformations to the face and brain, posing a significant impact on health care costs and the quality of life. METHODS: We have established a mouse line from a mutation that arose spontaneously in our wild-type colony that we called tuft. Tuft mice have heritable midline craniofacial defects featuring an anterior lipomatous cephalocele. RESULTS: Whole-mount skeletal stains indicated that affected newborns had a broader interfrontal suture where the cephalocele emerged between the frontal bones. Mice with a cephalocele positioned near the rostrum also presented craniofacial malformations such as ocular hypertelorism and midfacial cleft of the nose. Gross and histological examination revealed that the lipomatous cephalocele originated as a fluid filled cyst no earlier than E14.5 while embryos with a midfacial cleft was evident during craniofacial development at E11.5. Histological sections of embryos with a midfacial cleft revealed the cephalic neuroectoderm remained proximal or fused to the frontonasal ectoderm about the closure site of the anterior neuropore, indicating a defect to neural tube closure. We found the neural folds along the rostrum of E9 to E10.5 embryos curled inward and failed to close as well as embryos with exencephaly and anencephaly at later stages. Whole-mount in situ hybridization of anterior markers Fgf8 and Sonic hedgehog indicated closure of the rostral site was compromised in severe cases. CONCLUSION: We present a model demonstrating how anterior cranial cephaloceles are generated following a defect to neural tube closure and relevance to subsequent craniofacial morphogenesis in the tuft mouse.

How to form and close the brain: insight into the mechanism of cranial neural tube closure in mammals.

The development of the embryonic brain critically depends on successfully completing cranial neural tube closure (NTC). Failure to properly close the neural tube results in significant and potentially lethal neural tube defects (NTDs). We believe these malformations are caused by disruptions in normal developmental programs such as those involved in neural plate morphogenesis and patterning, tissue fusion, and coordinated cell behaviors. Cranial NTDs include anencephaly and craniorachischisis, both lethal human birth defects. Newly emerging methods for molecular and cellular analysis offer a deeper understanding of not only the developmental NTC program itself but also mechanical and kinetic aspects of closure that may contribute to cranial NTDs. Clarifying the underlying mechanisms involved in NTC and how they relate to the onset of specific NTDs in various experimental models may help us develop novel intervention strategies to prevent NTDs.

Stereological evaluation of the kidneys of anencephalic and normal fetuses.

The aim of this study is to test the glomerular and other quantitative parameters of kidneys of anencephalic fetuses and comparing those to "normal" fetuses. In this study, 20 kidneys of human fetuses (5 boys and 5 girls of anencephalic fetus, and 5 boys and 5 girls of normal fetus), at gestational ages of 25-30 weeks, were examined. This study is based on two basic research methods: one is a conventional anatomical measurement at the macroscopical level; the other is a design-biased stereological method at the microscopical level. Physical dissector and Cavalieri principle were used to estimate the total and numerical density of glomerulus and the volume of kidney, respectively. The results of the two types of investigation were compared based on anencephalic/normal and boy/girl kidneys at both the macroscopical and microscopical levels. There was no significant difference found between the quantitative features of kidneys (volume of kidneys and mean number and/or height of glomerulus) belonging to anencephalic and normal fetuses. The results of this study suggest that anencephalic fetuses did not differ from normal fetuses in respect of kidneys.

Brca1 deficiency results in early embryonic lethality characterized by neuroepithelial abnormalities.

The breast and ovarian cancer susceptibility gene, BRCA1, has been cloned and shown to encode a zinc-finger protein of unknown function. Mutations in BRCA1 account for at least 80% of families with both breast and ovarian cancer, as well as some non-familial sporadic ovarian cancers. The loss of wild-type BRCA1 in tumours of individuals carrying one nonfunctional BRCA1 allele suggests that BRCA1 encodes a tumour suppressor that may inhibit the proliferation of mammary epithelial cells. To examine the role of BRCA1 in normal tissue growth and differentiation, and to generate a potential model for the cancer susceptibility associated with loss of BRCA1 function, we have created a mouse line carrying a mutation in one Brca1 allele. Analysis of mice homozygous for the mutant allele indicate that Brca1 is critical for normal development, as these mice died in utero between 10 and 13 days of gestation (E10-E13). Abnormalities in Brca1-deficient embryos were most evident in the neural tube, with 40% of the embryos presenting with varying degrees of spina bifida and anencephaly. In addition, the neuroepithelium in Brca1-deficient embryos appeared disorganized, with signs of both rapid proliferation and excessive cell death.

[Fetal acardia in a twin pregnancy achieved by in vitro fertilization]. / Acardia fetal en embarazo gemelar logrado por fertilización in vitro.

The frequency of fetal arcadia in the world is 1:35,000 to 1:48,000 pregnancies; is currently estimated that 1% of newborns in developed countries are the result of assisted reproduction techniques and the frequency of twin pregnancies is close to 18%, of these, an estimated relative risk for cardiac defect is 1.6. However, the association of acardiac fetus, twin pregnancy and assisted reproductive techniques is not fully established. In this paper, we describe a case of fetal acardia, in a pregnancy resulting from assisted reproduction and its obstetric care.

Impaired retinal vascular development in anencephalic human fetus.

This study was to investigate the effect of the absence of ganglion cells on the development of human retinal vasculature. Anencephaly (AnC) and age-matched control eyes derived from each three spontaneously aborted fetus (ranging from 15 to 20 weeks gestation) were subjected to immunofluorescence staining for HIF-1alpha, Thy-1, glial fibrillary acidic protein (GFAP) and platelet/endothelial cell adhesion molecule (PECAM) and apoptosis assay. In developing mouse retina, Western blotting for hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) was performed. Under hypoxic condition (O(2) < 1%), cellular proliferation and VEGF mRNA expression in astrocytes were measured. Apoptotic cells in AnC retina were primarily localized in the ganglion cell layer (GCL), whereas apoptotic cells in normal retina were distributed in the retinoblastic layer. With increase of apoptotic cells in GCL of AnC retina, HIF-1alpha expression were severely distinguished in avascular retina and GFAP expression in junctional area between avascular and vascular retina was much reduced, accompanied by decrease of PECAM expression compared to normal retina. In developing mouse retina, HIF-1alpha and VEGF expression were high in hypoxic retina of early stage with incomplete vascular development and then progressively decreased with regression to arborous pattern of matured vascular networks. In hypoxic condition, a significant increase in cellular proliferation and VEGF mRNA expression was observed in astrocytes. Therefore, our results suggest that vascular attenuation in AnC retina could be closely related to the absence of ganglion cells as the metabolic demander to induce retinal vascular development.

Study of prostate growth in prune belly syndrome and anencephalic fetuses.

BACKGROUND: To compare the growth of the prostate in anencephalic, prune belly syndrome (PBS) and control fetuses. METHODS: We studied 35 prostates from normal human fetuses aged 11-22 weeks postconception (WPC); 15 from anencephalic fetuses aged 13-19 WPC; and 6 from PBS fetuses aged 13-31WPC. After prostate dissection, we evaluated the prostate length, width and thickness with the aid of a computer program (Image Pro and Image J). The fetal prostate volume (PV) was calculated using the ellipsoid formula: PV = [length × thickness × width] × 0.523. The prostates were dissected and the PV was measured with the aid of the same computer program. Means were statistically compared using the unpaired t-test and linear regression was performed. RESULTS: In 2 PBS fetuses we observed prostatic atresia. We did not observe significant differences in PV when comparing the control group (PV: 6.1 to 313.81 mm, mean = 70.85 mm: SD = 71.43 mm) with anencephalic fetuses: p = 0.3575 (PV: 5.1 to 159.11 mm, mean = 42.94 mm; SD = 40.11 mm) and PBS fetuses: p > 0.999 (PV: 10.89 to 148.71 mm, mean = 55.4 mm; SD = 63.64 mm). The linear regression analysis indicated that the PV in the control group (r2 = 0.3096; p = 0.0004), anencephalic group (r2 = 0.3778; p = 0.0148) and PBS group (r2 = 0.9821; p < 0.009) increased significantly and positively with fetal age (p < 0.0001). CONCLUSIONS: We did not observe significant differences in development of the prostate in fetuses with anencephaly and in 2/3 of fetuses with PBS during the fetal period studied. In 1/3 of the PBS fetuses, the prostate had important atresia. LEVEL OF EVIDENCE: Level III.

Epidemiology of acephalus/acardius monozygotic twins: new insights into an epigenetic causal hypothesis.

Apart from a series of 10 acephalus/acardius (Ac/Ac) cases described from a pathological point of view, and the analysis of a review of published cases, we have been unable to find any epidemiological studies on Ac/Ac. Using data from the Spanish Collaborative Study of Congenital Malformations (ECEMC), we present here what seems to be the first epidemiological analysis of a consecutive series of the Ac/Ac type of monozygotic twins (MZT). Among a total of 2,281,604 consecutive births, 11 cases of Ac/Ac MZT were detected, giving a frequency of 0.48 per 100,000 births. However, we consider the period 1980-1985 as the baseline for our data, as in this period voluntary termination of pregnancy was not possible in Spain, and the frequency of Ac/Ac MZT was 0.49 per 100,000 births. Nonetheless, this frequency should be considered as a minimal estimation. The characteristics of these Ac/Ac cases indicate that they are more frequent in males (sex ratio 2.67). In addition, gestational age in Ac/Ac cases was 2.41 and 3.12 weeks lower than in malformed and control twins, respectively. Similarly, their mothers are 4.54 and 4.68 years younger than mothers of separate malformed and control twins, respectively. To understand the biological basis behind the occurrence of MZT in the context of recent observations, we evaluate the hypothesis that the epigenetic processes involved in the early cleavage of the embryo, and in blastocyst formation during development, may be implicated in twinning.

Acephalous lamb from an in vitro-produced sheep embryo.

This is the first report of an acephalous lamb from the transfer of an in vitro-produced sheep embryo. Twelve in vitro-fertilized embryos were transferred to 4 recipient ewes (3 embryos/ewe). Two ewes remained pregnant: one delivered a normal female lamb, the other a male acephalous lamb. Possible contributing factors are discussed.

Cell necrosis, intrinsic apoptosis and senescence contribute to the progression of exencephaly to anencephaly in a mice model of congenital chranioschisis.

Exencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord.

Abnormal folate metabolism in foetuses affected by neural tube defects.

Folic acid supplementation can prevent many cases of neural tube defects (NTDs), whereas suboptimal maternal folate status is a risk factor, suggesting that folate metabolism is a key determinant of susceptibility to NTDs. Despite extensive genetic analysis of folate cycle enzymes, and quantification of metabolites in maternal blood, neither the protective mechanism nor the relationship between maternal folate status and susceptibility are understood in most cases. In order to investigate potential abnormalities in folate metabolism in the embryo itself, we derived primary fibroblastic cell lines from foetuses affected by NTDs and subjected them to the dU suppression test, a sensitive metabolic test of folate metabolism. Significantly, a subset of NTD cases exhibited low scores in this test, indicative of abnormalities in folate cycling that may be causally linked to the defect. Susceptibility to NTDs may be increased by suppression of the methylation cycle, which is interlinked with the folate cycle. However, reduced efficacy in the dU suppression test was not associated with altered abundance of the methylation cycle intermediates, s-adenosylmethionine and s-adenosylhomocysteine, suggesting that a methylation cycle defect is unlikely to be responsible for the observed abnormality of folate metabolism. Genotyping of samples for known polymorphisms in genes encoding folate-associated enzymes did not reveal any correlation between specific genotypes and the observed abnormalities in folate metabolism. These data suggest that as yet unrecognized genetic variants result in embryonic abnormalities of folate cycling that may be causally related to NTDs.

Molecular cytogenetic characterization of a duplication of 15q24.2-q26.2 associated with anencephaly and neural tube defect.

OBJECTIVE: We present molecular cytogenetic characterization of a duplication of 15q24.2-q26.2 associated with anencephaly and neural tube defect (NTD). CASE REPORT: A 35-year-old pregnant woman was found to have a fetus with anencephaly by prenatal ultrasound at 12 weeks of gestation. The pregnancy was subsequently terminated, and a malformed fetus was delivered with anencephaly. Cytogenetic analysis of the cultured placental tissues revealed a karyotype of 46,XX,dup(15) (q24.2q26.2). Parental karyotypes were normal. Array comparative genomic hybridization analysis of the placental tissues revealed a 20.36-Mb duplication of 15q24.2-q26.2 encompassing 100 Online Mendelian Inheritance of in Man (OMIM) genes including LINGO1, MTHFS, KIF7 and CHD2. Metaphase fluorescence in situ hybridization analysis using 15q25.1-specidic probe confirmed a duplication of 15q25.1. Polymorphic DNA marker analysis showed a maternal origin of the duplication. CONCLUSION: A duplication of chromosome 15q24.2-q26.2 can be associated with NTD.

Apoptosis, Expression of PAX3 and P53, and Caspase Signal in Fetuses with Neural Tube Defects.

BACKGROUND: Neural tube defects (NTDs) are among the most common and severe congenital malformations of the central nervous system. Animal studies have shown that apoptosis is involved in the development of NTDs. However, little evidence is available from human studies. We aim to examine the level of apoptosis and expression of apoptosis-regulating proteins of human terminated fetuses. METHODS: A total of 37 NTD cases and 21 controls from pregnancy terminations were recruited. Tissues of the central nervous system were obtained through autopsy. Apoptosis of neuroepithelial cells was examined by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) assay. Expression of PAX3, p53, and caspase 3/8/9 in central nervous tissue was measured using Western blotting. RESULTS: More TUNEL-positive apoptosis cells were observed in the central nervous tissue of NTD cases than those of controls (p < 0.05). In spinal cord tissue, lower PAX3 expression, higher p53 expression, and increased levels of cleaved caspase 3(17kD) and cleaved caspase 8 (18kD) were found in anencephaly cases but not in spina bifida cases when compared with controls. In brain tissue, levels of PAX3 were significantly reduced in both encephalocele and spina bifida subtypes; the expression levels of cleaved caspase 3(17 kD) of encephalocele cases and cleaved caspase 8(47/45 kD) in spina bifida cases were higher than in controls; no difference was found in the expression of p53 or caspase 9 between NTDs and controls. CONCLUSION: These findings provide some evidence that excessive apoptosis in fetal central nervous tissues may be associated with the development of human NTDs. Birth Defects Research 109:1596-1604, 2017. © 2017 Wiley Periodicals, Inc.

A case report of recurrent anencephaly and literature review.

Anencephaly is a rare congenital anomaly in which the forebrain, meninges, vault of the skull, and scalp all fail to form. We report a case of a 32-year-old gravida 2 woman with an anencephalic fetus detected at the 21st gestational week. She had a history of an intrauterine fetal death of an anencephalic fetus at the 20th gestational week two years before. We present the case and briefly review the literature.

Brain tissue fragments in the amniotic fluid of rats with neural tube defect.

AIMS: Brain tissue nodules are occasionally seen in the lungs of neural tube defect (NTD) cases. We looked for brain tissue fragments in amniotic fluid of rats with NTD as it is the basis for the aspiration hypothesis. METHODS: Eighty-seven pregnant rats were randomly divided into experimental (n=58) and control (n=29) groups. Experimental rats received 100,000 U of vitamin A in 1 mL of corn oil on gestational days 8, 9 and 10, while control rats received corn oil. On gestational days 15, 18, 19, 20 and 21, amniotic fluid was drawn from three control animals and five experimental animals and analysed. RESULTS: NTD was found in 22.75% of experimental fetuses and in no control fetuses. Brain tissue fragment number and volume fraction increased between gestational days 18 and 20, falling on day 21. CONCLUSIONS: Excessive doses of vitamin A induce a high rate of early fetal death and development of NTD. Brain tissue fragments in the amniotic fluid reflect the evolution from exencephaly to anencephaly and could support the aspiration hypothesis. However, as it is a late event in the rat, this model may not reproduce the brain tissue nodules in the lung.

Acardiac acephalic twin gestation with transposition of great arteries in pump twin.

We report here a case of twin reversed arterial perfusion sequence with transposition of great arteries in the pump twin. Color Doppler and fetal echocardiographic examination revealed an acardiac, anencephalic twin without upper limbs and a pump twin with transposition of great arteries. To our knowledge despite multiple anomalies reported in the acardiac twin, major anomalies, especially of a cardiac type are extremely rare and not reported in the pump twin, which maintains circulation. Management is directed at saving the pump fetus; therefore, we believe that during sonographic exam, emphasis should be placed on associated organ anomalies or complications that may hamper viability of the pump.

Generation of anencephaly: 1. Aberrant neurulation and 2. Conversion of exencephaly to anencephaly.

An experimental model for anencephaly was used to focus on two important aspects of the development of anencephaly: neurulation and conversion of exencephaly to anencephaly. Vitamin A was administered to pregnant rats on gestational days nine and ten. The animals were killed on successive gestational days to allow study of the development of anencephaly. The scanning electron microscope revealed filopodia and lamellopodia as the predominant mode of initial neural fold contact in the controls. Intertwining and overlapping of filopodia and lamellopodia with fusion of the adjacent cutaneous ectoderm completed neurulation. In embryos developing anencephaly, filopodia and lamellopodia never made contact above the cervical region and exencephaly resulted. The first evidence of the conversion of exencephaly to anencephaly was profound, labyrinthine expansion of the extracellular space of the telencephalic mantle. In spite of normal vascular patency and intact vessel walls, the exencephalic malformation spontaneously disintegrated, converting the lesion to anencephaly. The causes for tissue disintegration other than infarction must be considered in reconstructing the pathogenesis of anencephaly.