Regional neural tube closure defined by the Grainy head-like transcription factors

Primary neurulation in mammals has been defined by distinct anatomical closure sites, at the hindbrain/cervical spine (closure 1), forebrain/midbrain boundary (closure 2), and rostral end of the forebrain (closure 3). Zones of neurulation have also been characterized by morphologic differences in neural fold elevation, with non-neural ectoderm-induced formation of paired dorso-lateral hinge points (DLHP) essential for neural tube closure in the cranial and lower spinal cord regions, and notochord-induced bending at the median hinge point (MHP) sufficient for closure in the upper spinal region. Here we identify a unifying molecular basis for these observations based on the function of the non-neural ectoderm-specific Grainy head-like genes in mice. Using a gene-targeting approach we show that deletion of Grhl2 results in failed closure 3, with mutants exhibiting a split-face malformation and exencephaly, associated with failure of neuro-epithelial folding at the DLHP. Loss of Grhl3 alone defines a distinct lower spinal closure defect, also with defective DLHP formation. The two genes contribute equally to closure 2, where only Grhl gene dosage is limiting. Combined deletion of Grhl2 and Grhl3 induces severe rostral and caudal neural tube defects, but DLHP-independent closure 1 proceeds normally in the upper spinal region. These findings provide a molecular basis for non-neural ectoderm mediated formation of the DLHP that is critical for complete neuraxis closure.     

Integrity of the methylation cycle is essential for mammalian neural tube closure

BACKGROUND: Closure of the cranial neural tube during embryogenesis is a crucial process in development of the brain. Failure of this event results in the severe neural tube defect (NTD) exencephaly, the developmental forerunner of anencephaly. METHODS: The requirement for methylation cycle function in cranial neural tube closure was tested by treatment of cultured mouse embryos with cycloleucine or ethionine, inhibitors of methionine adenosyl transferase. Embryonic phenotypes were investigated by histological analysis, and immunostaining was performed for markers of proliferation and apoptosis. Methylation cycle intermediates s-adenosylmethionine and s-adenosylhomocysteine were also quantitated by tandem mass spectrometry. RESULTS: Ethionine and cycloleucine treatments significantly reduced the ratio of abundance of s-adenosylmethionine to s-adenosylhomocysteine and are, therefore, predicted to suppress the methylation cycle. Exposure to these inhibitors during the period of cranial neurulation caused a high incidence of exencephaly, in the absence of generalized toxicity, growth retardation, or developmental delay. Reduced neuroepithelial thickness and reduced density of cranial mesenchyme were detected in ethionine-treated but not cycloleucine-treated embryos that developed exencephaly. Reduced mesenchymal density is a potential cause of ethionine-induced exencephaly, although we could not detect a causative alteration in proliferation or apoptosis prior to failure of neural tube closure. CONCLUSIONS: Adequate functioning of the methylation cycle is essential for cranial neural tube closure in the mouse, suggesting that suppression of the methylation cycle could also increase the risk of human NTDs. We hypothesize that inhibition of the methylation cycle causes NTDs due to disruption of crucial reactions involving methylation of DNA, proteins or other biomolecules.     

The actin depolymerizing factor n-cofilin is essential for neural tube morphogenesis and neural crest cell migration

Cofilin/ADF proteins are a ubiquitously expressed family of F-actin depolymerizing factors found in eukaryotic cells including plants. In vitro, cofilin/ADF activity has been shown to be essential for actin driven motility, by accelerating actin filament turnover. Three actin depolymerizing factors (n-cofilin, m-cofilin, ADF) can be found in mouse and human. Here we show that in mouse the non-muscle-specific gene-n-cofilin-is essential for migration of neural crest cells as well as other cell types in the paraxial mesoderm. The main defects observed in n-cofilin mutant embryos are an impaired delamination and migration of neural crest cells, affecting the development of neural crest derived tissues. Neural crest cells lacking n-cofilin do not polarize, and F-actin bundles or fibers are not detectable. In addition, n-cofilin is required for neuronal precursor cell proliferation and scattering. These defects result in a complete lack of neural tube closure in n-cofilin mutant embryos. Although ADF is overexpressed in mutant embryos, this cannot compensate the lack of n-cofilin, suggesting that they might have a different function in embryonic development. Our data suggest that in mammalian development, regulation of the actin cytoskeleton by the F-actin depolymerizing factor n-cofilin is critical for epithelial-mesenchymal type of cell shape changes as well as cell proliferation.     

Arx homeobox gene is essential for development of mouse olfactory system

The olfactory system provides an excellent model in which to study cell proliferation, migration, differentiation, axon guidance, dendritic morphogenesis, and synapse formation. We report here crucial roles of the Arx homeobox gene in the developing olfactory system by analyzing its mutant phenotypes. Arx protein was expressed strongly in the interneurons and weakly in the radial glia of the olfactory bulb, but in neither the olfactory sensory neurons nor bulbar projection neurons. Arx-deficient mice showed severe anatomical abnormalities in the developing olfactory system: (1) size reduction of the olfactory bulb, (2) reduced proliferation and impaired entry into the olfactory bulb of interneuron progenitors, (3) loss of tyrosine hydroxylase-positive periglomerular cells, (4) disorganization of the layer structure of the olfactory bulb, and (5) abnormal axonal termination of olfactory sensory neurons in an unusual axon-tangled structure, the fibrocellular mass. Thus, Arx is required for not only the proper developmental processes of Arx-expressing interneurons, but also the establishment of functional olfactory neural circuitry by affecting Arx-non-expressing sensory neurons and projection neurons. These findings suggest a likely role of Arx in regulating the expression of putative instructive signals produced in the olfactory bulb for the proper innervation of olfactory sensory axons.     

Clusterin gene transcription is activated by caudal-related homeobox genes in intestinal epithelium

Caudal-related homeobox (Cdx) proteins play an important role in development and differentiation of the intestinal epithelium. Using cDNA differential display, we identified clusterin as a prominently induced gene in a Cdx2-regulated cellular model of intestinal differentiation. Transfection experiments and DNA-protein interaction assays showed that clusterin is an immediate downstream target gene for Cdx proteins. The distribution of clusterin protein in the intestine was assessed during development and in the adult epithelium using immunohistochemistry. In the adult mouse epithelium, clusterin protein was localized in both crypt and villus compartments but not in interstitial cells of the intestinal mucosa. Together, these data suggest that clusterin is a direct target gene for Cdx homeobox proteins, and the pattern of clusterin protein expression suggests that it is associated with the differentiated state in the intestinal epithelium.     

Genetic interaction between the homeobox transcription factors HESX1 and SIX3 is required for normal pituitary development

Hesx1 has been shown to be essential for normal pituitary development. The homeobox gene Six3 is expressed in the developing pituitary gland during mouse development but its function in this tissue has been precluded by the fact that in the Six3-deficient embryos the pituitary gland is not induced. To gain insights into the function of Six3 during pituitary development we have generated Six3^+/−;Hesx1^Cre/+ double heterozygous mice. Strikingly, these mice show marked dwarfism, which is first detectable around weaning, and die by the 5th-6th week of age. Thyroid and gonad development is also impaired in these animals. Analysis of Six3^+/−;Hesx1^Cre/+ compound embryos indicates that hypopituitarism is the likely cause of these defects since pituitary development is severely impaired in these mutants. Similar to the Hesx1-deficient embryos, Rathke's pouch is initially expanded in Six3^+/−;Hesx1^Cre/+ compound embryos due to an increase in cell proliferation. Subsequently, the anterior pituitary gland appears bifurcated, dysmorphic and occasionally ectopically misplaced in the nasopharyngeal cavity, but cell differentiation is unaffected. Our research has revealed a role for Six3 in normal pituitary development, which has likely been conserved during evolution as SIX3 is also expressed in the pituitary gland of the human embryo.     

A brain-specific homeobox gene, Bsx, is essential for proper postnatal growth and nursing

To investigate in vivo roles of a murine hypothalamic homeobox gene, Bsx, we generated and analyzed two mutant alleles, Bsx(DeltaHD) and Bsx(lacZ). Bsx(DeltaHD) lacks the homeodomain, and Bsx(lacZ) is an insertion of a lacZ reporter gene. Bsx-lacZ expression was detected in the hypothalamus and pineal gland and reiterates Bsx expression. Bsx homozygous mutant mice were born at the expected Mendelian ratio, but their growth was impaired. Offspring from Bsx homozygous mutant females exhibited a low survival rate due to a nursing defect. Mammary glands of the mutant females developed normally during pregnancy; however, they involuted quickly after parturition. These results demonstrate that Bsx is required for postnatal growth and maintenance of lactating mammary glands. Thus, mouse Bsx is likely involved in systemic control of suppression of apoptosis of postpartum mammary epithelial cells.     

Dishevelled 2 is essential for cardiac outflow tract development,somite segmentation and neural tube closure

The murine dishevelled 2 (Dvl2) gene is an ortholog of the Drosophila segment polarity gene Dishevelled, a member of the highly conserved Wingless/Wnt developmental pathway. Dvl2-deficient mice were produced to determine the role of Dvl2 in mammalian development. Mice containing null mutations in Dvl2 present with 50% lethality in both inbred 129S6 and in a hybrid 129S6-NIH Black Swiss background because of severe cardiovascular outflow tract defects, including double outlet right ventricle, transposition of the great arteries and persistent truncus arteriosis. The majority of the surviving Dvl2(-/-) mice were female, suggesting that penetrance was influenced by sex. Expression of Pitx2 and plexin A2 was attenuated in Dvl2 null mutants, suggesting a defect in cardiac neural crest development during outflow tract formation. In addition, approximately 90% of Dvl2(-/-) mice have vertebral and rib malformations that affect the proximal as well as the distal parts of the ribs. These skeletal abnormalities were more pronounced in mice deficient for both Dvl1 and Dvl2. Somite differentiation markers used to analyze Dvl2(-/-) and Dvl1(-/-);Dvl2(-/-) mutant embryos revealed mildly aberrant expression of Uncx4.1, delta 1 and myogenin, suggesting defects in somite segmentation. Finally, 2-3% of Dvl2(-/-) embryos displayed thoracic spina bifida, while virtually all Dvl1/2 double mutant embryos displayed craniorachishisis, a completely open neural tube from the midbrain to the tail. Thus, Dvl2 is essential for normal cardiac morphogenesis, somite segmentation and neural tube closure, and there is functional redundancy between Dvl1 and Dvl2 in some phenotypes.     

Actin polymerization is essential for pollen tube growth

Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a approximately 50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a approximately 100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1--4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming.     

E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions

Cadherin adhesion molecules are key determinants of morphogenesis and tissue architecture. Nevertheless, the molecular mechanisms responsible for the morphogenetic contributions of cadherins remain poorly understood in vivo. Besides supporting cell-cell adhesion, cadherins can affect a wide range of cellular functions that include activation of cell signalling pathways, regulation of the cytoskeleton and control of cell polarity. To determine the role of E-cadherin in stratified epithelium of the epidermis, we have conditionally inactivated its gene in mice. Here we show that loss of E-cadherin in the epidermis in vivo results in perinatal death of mice due to the inability to retain a functional epidermal water barrier. Absence of E-cadherin leads to improper localization of key tight junctional proteins, resulting in permeable tight junctions and thus altered epidermal resistance. In addition, both Rac and activated atypical PKC, crucial for tight junction formation, are mislocalized. Surprisingly, our results indicate that E-cadherin is specifically required for tight junction, but not desmosome, formation and this appears to involve signalling rather than cell contact formation.     

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.