Development and tissue origins of the mammalian cranial base.

The vertebrate cranial base is a complex structure composed of bone, cartilage and other connective tissues underlying the brain; it is intimately connected with development of the face and cranial vault. Despite its central importance in craniofacial development, morphogenesis and tissue origins of the cranial base have not been studied in detail in the mouse, an important model organism. We describe here the location and time of appearance of the cartilages of the chondrocranium. We also examine the tissue origins of the mouse cranial base using a neural crest cell lineage cell marker, Wnt1-Cre/R26R, and a mesoderm lineage cell marker, Mesp1-Cre/R26R. The chondrocranium develops between E11 and E16 in the mouse, beginning with development of the caudal (occipital) chondrocranium, followed by chondrogenesis rostrally to form the nasal capsule, and finally fusion of these two parts via the midline central stem and the lateral struts of the vault cartilages. X-Gal staining of transgenic mice from E8.0 to 10 days post-natal showed that neural crest cells contribute to all of the cartilages that form the ethmoid, presphenoid, and basisphenoid bones with the exception of the hypochiasmatic cartilages. The basioccipital bone and non-squamous parts of the temporal bones are mesoderm derived. Therefore the prechordal head is mostly composed of neural crest-derived tissues, as predicted by the New Head Hypothesis. However, the anterior location of the mesoderm-derived hypochiasmatic cartilages, which are closely linked with the extra-ocular muscles, suggests that some tissues associated with the visual apparatus may have evolved independently of the rest of the "New Head".

Effects of the curly tail genotype on neuroepithelial integrity and cell proliferation during late stages of primary neurulation.

The curly tail (ct/ct) mouse mutant shows a high frequency of delay or failure of neural tube closure, and is a good model for human neural tube defects, particularly spina bifida. In a previous study we defined distinct domains of gene expression in the caudal region of non-mutant embryos during posterior (caudal) neuropore closure (Gofflot et al. Developmental Dynamics 210, 431-445, 1997). Here we use BrdU incorporation into S-phase nuclei to investigate the relationship between cell proliferation and the previously described gene expression domains in ct/ct mutant embryos. The BrdU-immunostained sections were also examined for abnormalities of tissue structure; immunohistochemical detection of perlecan (an extracellular heparan sulphate proteoglycan) was used as an indicator of neuroepithelial basement membrane structure and function. Quantitation of BrdU uptake revealed that at early stages of neurulation, cell proliferation was specifically reduced in the paraxial mesoderm of all ct/ct embryos compared with wild type controls, but at later stages (more cranial levels) it was increased. Those ct/ct embryos with enlarged posterior neuropore (indicating delay of closure) additionally showed an increased BrdU labelling index within the open neuroepithelium at all axial levels; however, this tissue was highly abnormal with respect to cell and nuclear morphology. It showed cell death and loss of cells from the apical surface, basement membrane defects including increased perlecan immunoreactivity, and increased separation from the underlying mesenchyme and notochord. These observations suggest that the mechanism of delay or failure of neuroepithelial curvature that leads to neural tube defects in curly tail embryos involves abnormalities of neuroepithelial-mesenchymal interactions that may be initiated by abnormal cellular function within the neuroepithelium. Minor histological and proliferation abnormalities are present in all ct/ct embryos, regardless of phenotype.

De novo alu-element insertions in FGFR2 identify a distinct pathological basis for Apert syndrome.

Apert syndrome, one of five craniosynostosis syndromes caused by allelic mutations of fibroblast growth-factor receptor 2 (FGFR2), is characterized by symmetrical bony syndactyly of the hands and feet. We have analyzed 260 unrelated patients, all but 2 of whom have missense mutations in exon 7, which affect a dipeptide in the linker region between the second and third immunoglobulin-like domains. Hence, the molecular mechanism of Apert syndrome is exquisitely specific. FGFR2 mutations in the remaining two patients are distinct in position and nature. Surprisingly, each patient harbors an Alu-element insertion of approximately 360 bp, in one case just upstream of exon 9 and in the other case within exon 9 itself. The insertions are likely to be pathological, because they have arisen de novo; in both cases this occurred on the paternal chromosome. FGFR2 is present in alternatively spliced isoforms characterized by either the IIIb (exon 8) or IIIc (exon 9) domains (keratinocyte growth-factor receptor [KGFR] and bacterially expressed kinase, respectively), which are differentially expressed in mouse limbs on embryonic day 13. Splicing of exon 9 was examined in RNA extracted from fibroblasts and keratinocytes from one patient with an Alu insertion and two patients with Pfeiffer syndrome who had nucleotide substitutions of the exon 9 acceptor splice site. Ectopic expression of KGFR in the fibroblast lines correlated with the severity of limb abnormalities. This provides the first genetic evidence that signaling through KGFR causes syndactyly in Apert syndrome.

Genetic patterning of the posterior neuropore region of curly tail mouse embryos: deficiency of Wnt5a expression.

The mouse mutant curly tail (ct) develops tail flexion defects and spina bifida as the result of delayed or failed closure of the posterior neuropore (PNP). With the aim of identifying genes involved in the chain of events resulting in defective neurulation, which can be detected at day 10.5 of development, we examined the expression patterns of a number of genes implicated in patterning of axial structures, mesoderm and neuroepithelium. The genes analyzed were Shh, HNF3alpha, HNF3beta, Brachyury, Hoxb1, Evx1, Fgf8, Wnt5a and Wnt5b. No differences could be detected between non-mutant embryos and ct/ct embryos with normal PNP size for any of these genes. Comparisons between ct/ct embryos with enlarged PNP and phenotypically normal ct/ct or nonmutant embryos showed differences only for Wnt5a. Expression of this gene was greatly reduced in the ventral caudal mesoderm and hindgut endoderm. Analysis of younger embryos revealed that prior to the stage at which embryos at risk of developing neural tube defects can be detected, the same proportion of ct/ct embryos shows reduced Wnt5a expression. The proportion of embryos showing reduced expression and almost undetectable expression of Wnt5a reflects the proportions of tail defects and spina bifida seen at later stages. We suggest that deficiency of Wnt5a signaling in the ventral caudal region tissues is an important component of the mechanism of development of the defects in affected curly tail mutant mice, and that it is causally related to decreased cell proliferation within the ventral caudal region. A possible relationship between decreased Wnt5a expression and reduced levels of heparan sulphate proteoglycan is discussed.

Sonic hedgehog is not required for polarising activity in the Doublefoot mutant mouse limb bud.

The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly of all four limbs. We have analysed limb development in this mutant with respect to morphogenesis, gene expression patterns and ectopic polarising activity. The results reveal a gain-of-function mutation at a locus that mediates pattern formation in the developing limb. Shh expression is identical with that of wild-type embryos, i.e. there is no ectopic expression. However, mesenchyme from the anterior aspects of Dbf/+ mutant limb buds, when transplanted to the anterior side of chick wing buds, induces duplication of the distal skeletal elements. Mid-distal mesenchymal transplants from early, but not later, Dbf/+ limb buds are also able to induce duplication. This demonstration of polarising activity in the absence of Shh expression identifies the gene at the Dbf locus as a new genetic component of the Shh signalling pathway, which (at least in its mutated form) is able to activate signal transduction independently of Shh. The mutant gene product is sufficient to fulfil the signalling properties of Shh including upregulation of the direct Shh target genes Ptc and Gli, and induction of the downstream target genes Bmp2, Fgf4 and Hoxd13. The expression domains of all these genes extend from their normal posterior domains into the anterior part of the limb bud without being focused on a discrete ectopic site. These observations dissociate polarising activity from Shh gene expression in the Dbf/+ limb bud. We suggest that the product of the normal Dbf gene is a key active constituent of the polarising region, possibly acting in the extracellular compartment.

Fgfr2 and osteopontin domains in the developing skull vault are mutually exclusive and can be altered by locally applied FGF2.

Mutations in the human fibroblast growth factor receptor type 2 (FGFR2) gene cause craniosynostosis, particularly affecting the coronal suture. We show here that, in the fetal mouse skull vault, Fgfr2 transcripts are most abundant at the periphery of the membrane bones; they are mutually exclusive with those of osteopontin (an early marker of osteogenic differentiation) but coincide with sites of rapid cell proliferation. Fibroblast growth factor type 2 (FGF2) protein, which has a high affinity for the FGFR2 splice variant associated with craniosynostosis, is locally abundant; immunohistochemical detection showed it to be present at low levels in Fgfr2 expression domains and at high levels in differentiated areas. Implantation of FGF2-soaked beads onto the fetal coronal suture by ex utero surgery resulted in ectopic osteopontin expression, encircled by Fgfr2 expression, after 48 hours. We suggest that increased FGF/FGFR signalling in the developing skull, whether due to FGFR2 mutation or to ectopic FGF2, shifts the cell proliferation/differentiation balance towards differentiation by enhancing the normal paracrine down-regulation of Fgfr2.

CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis.

CD34 is a cell surface glycoprotein that is selectively expressed within the human hematopoietic system on stem and progenitor cells, and in early blood vessels. To elucidate its functions during early blood vessel formation and hematopoiesis, we analyzed the expression patterns, in day 8 to day 10 mouse embryos, of CD34 RNA by in situ hybridization and protein by immunohistochemistry using the monclonal antibody RAM 34. Levels of expression in embryonic blood vessels were correlated with the mode of vessel formation, being high in pre-endothelial cells and in vessels forming by vasculogenesis (particularly the dorsal aortae) or angiogenesis, but low in vessels forming by coalescence (the cardinal veins). CD34+ erythroid cells, presumably of yolk sac origin, were present in the liver of day 10 embryos; at the same stage, putative definitive hematopoietic cells, strongly CD34+, were present in the para-aortic mesenchyme. Possible sites of hemangioblastic differentiation were detected in the form of CD34+ endothelium-attached hematopoietic cells in the dorsal aorta and in two previously unreported locations, the proximal umbilical and vitelline arteries. These observations suggest functions for CD34 in relation to specific modes of blood vessel formation, and a hemangioblastic role in both embryonic and extraembryonic sites.

Apoptotic cell death in neuronal differentiation of P19 EC cells: cell death follows reentry into S phase.

Apoptotic cell death was observed during aggregate culture of the mouse embryonal carcinoma cell line P19 exposed to all-trans retinoic acid (tRA). This finding was confirmed by genomic DNA agarose gel electrophoresis and transmission electron microscopy. Apoptosis was associated with P19 cell neuronal differentiation; alternative causes of cell death, i.e., cavitation-related, cytotoxicity of tRA, or spontaneous cell death were excluded. Analysis by flow cytometry revealed that the apoptosis was likely to occur in multiplying cells that underwent to reentering into S phase. We therefore examined 5-bromo-2'-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression and localization in the aggregates by immunofluorescent staining. Although the P19 cells in the aggregates exposed to tRA incorporated BrdU at an equivalent level to those not exposed to tRA, the cells showed diminished PCNA expression and nuclear accumulation. We propose that P19 apoptosis during neuronal differentiation is a model system in which programmed cell death occurs simultaneously with cell division leading to differentiation.

Genetic patterning of the developing mouse tail at the time of posterior neuropore closure.

Posterior neuropore (PNP) closure coincides with the end of gastrulation, marking the end of primary neurulation and primary body axis formation. Secondary neurulation and axis formation involve differentiation of the tail bud mesenchyme. Genetic control of the primary-secondary transition is not understood. We report a detailed analysis of gene expression in the caudal region of day 10 mouse embryos during primary neuropore closure. Embryos were collected at the 27-32 somite stage, fixed, processed for whole mount in situ hybridisation, and subsequently sectioned for a more detailed analysis. Genes selected for study include those involved in the key events of gastrulation and neurulation at earlier stages and more cranial levels. Patterns of expression within the tail bud, neural plate, recently closed neural tube, notochord, hindgut, mesoderm, and surface ectoderm are illustrated and described. Specifically, we report continuity of expression of the genes Wnt5a, Wnt5b, Evx1, Fgf8, RARgamma, Brachyury, and Hoxb1 from primitive streak and node into subpopulations of the tail bud and caudal axial structures. Within the caudal notochord, developing floorplate, and hindgut, HNF3alpha, HNF3beta, Shh, and Brachyury expression domains correlate directly with known genetic roles and predicted tissue interdependence during induction and differentiation of these structures. The patterns of expression of Wnt5a, Hoxb1, Brachyury, RARgamma, and Evx1, together with observations on proliferation, reveal that the caudal mesoderm is organised at a molecular level into distinct domains delineated by longitudinal and transverse borders before histological differentiation. Expression of Wnt5a in the ventral ectodermal ridge supports previous evidence that this structure is involved in epithelial-mesenchymal interaction. These results provide a foundation for understanding the mechanisms facilitating transition from primary to secondary body axis formation, as well as the factors involved in defective spinal neurulation.

Expression of Fgf-3 in relation to hindbrain segmentation, otic pit position and pharyngeal arch morphology in normal and retinoic acid-exposed mouse embryos.

The gene Fgf-3 is expressed in rhombomeres 5 and 6 of the hindbrain and has been functionally implicated in otic development. We describe new sites of expression of this gene in mouse embryos in the forebrain, the midbrain-hindbrain junction region, rhombomere boundaries, a cranial surface ectodermal domain that includes the otic placode, and in the most recently formed somite. In the early hindbrain, high levels of Fgf-3 transcripts are present in rhombomere 4. The surface ectodermal domain at first (day 8 1/2) extends laterally from rhombomeres 4 and 5 (prorhombomere B), in which neuroepithelial levels of expression are highest, to the second pharyngeal arch ventrally; at day 9, when the region of highest level of neuroepithelial Fgf-3 expression is in rhombomeres 5 and 6, the dorsal origin of the surface ectodermal domain is also at this level, extending obliquely to the otic placode and the second arch. The initially high level of Fgf-3 transcripts in the otic placode is downregulated as the placode invaginates to form the otic pit. Fgf-3 is a good marker for the epithelium of pharyngeal arches 2 and 3, and our in situ hybridization results confirm the dual identity of the apparently fused first and second arches in some retinoic acid-exposed embryos, and the fusion of the first arch with the maxillary region in others. Correlation between Fgf-3 expression and morphological pattern in craniofacial tissues of normal and retinoic acid-exposed embryos indicates that prorhombomere B, the second arch and the otic ectoderm represent a cranial segment whose structural integrity is maintained when hindbrain morphology and pharyngeal arch morphology are altered. Comparison of normal Fgf-3 expression domains with those of Fgf-4 and with the phenotype of Fgf-3-deficient mutant embryos suggests that there is some functional redundancy between Fgf-3 and Fgf-4 in otic induction and second arch development.

The effects of retinoid status on TGF beta expression during mouse embryogenesis.

In a previous study we investigated the effects of RA excess on TGF beta protein localization in early postimplantation stages of mouse development. Here we extend this investigation by comparing the effects of retinoid deficiency with those of excess, and by comparing the effects of altered retinoid status on TGF beta protein and RNA transcript distribution. In vitamin A-deficient embryos, TGF beta 1 RNA and protein distribution were both unaltered compared with controls; conversely, TGF beta 2 protein levels were reduced while RNA levels remained normal. In RA-treated embryos, the previous study showed that intracellular TGF beta 1 levels were decreased, while those of extracellular TFG beta 1 were initially decreased but subsequently increased; here we found that TGF beta 1 RNA transcript levels were reduced following exposure to RA excess. TGF beta 2 showed a clear disparity between the effects of RA excess on protein and RNA transcript levels: RNA transcript distribution was unchanged or showed a slight increase in RA-treated embryos, whereas the previous results showed greatly reduced protein levels. The new results provide further evidence for interaction between retinoids and TGF beta s during mouse development, and indicate that retinoids are capable of differentially regulating TGF beta isoforms through mechanisms involving different stages in the process of TGF beta synthesis and secretion. The long-term nature of the effects of transient exposure to RA excess suggests that the mechanisms of RA-TGF beta interaction may be indirect.

Genesis and prevention of spinal neural tube defects in the curly tail mutant mouse: involvement of retinoic acid and its nuclear receptors RAR-beta and RAR-gamma.

A role for all-trans-retinoic acid in spinal neurulation is suggested by: (1) the reciprocal domains of expression of the retinoic acid receptors RAR-beta and RAR-gamma in the region of the closed neural tube and open posterior neuropore, respectively, and (2) the preventive effect of maternally administered retinoic acid (5 mg/kg) on spinal neural tube defects in curly tail (ct/ct) mice. Using in situ hybridisation and computerised image analysis we show here that in ct/ct embryos, RAR-beta transcripts are deficient in the hindgut endoderm, a tissue whose proliferation rate is abnormal in the ct mutant, and RAR-gamma transcripts are deficient in the tail bud and posterior neuropore region. The degree of deficiency of RAR-gamma transcripts is correlated with the severity of delay of posterior neuropore closure. As early as 2 hours following RA treatment at 10 days 8 hours post coitum, i.e. well before any morphogenetic effects are detectable, RAR-beta expression is specifically upregulated in the hindgut endoderm, and the abnormal expression pattern of RAR-gamma is also altered. These results suggest that the spinal neural tube defects which characterise the curly tail phenotype may be due to interaction between the ct gene product and one or more aspects of the retinoic acid signalling pathway.

Analysis of limb reduction defects in babies exposed to chorionic villus sampling.

In 1991 we reported a cluster of babies with limb abnormalities and suggested that chorionic villus sampling (CVS) was aetiologically associated with these defects. To address the issue more objectively, we have assessed reported limb reduction defects in 75 babies exposed to CVS in utero. 13 babies had an absent limb or a defect through the humerus or femur; 9 had defects through the radius or tibia; 22 defects of the carpus, tarsus, metacarpus, or metatarsus; 25 defects of the digits; and 6 defects of the terminal phalanx or nail only. There was a strong correlation between the severity of the defects and the duration of gestation when CVS was done. The median gestational age at CVS ranged from 56 (range 49-65) postmenstrual days for the most severe category to 72 (51-98) days for the least severe. The relation was seen for both isolated limb defects and for cases with oromandibular-limb hypogenesis syndromes. This relation is further evidence that CVS has an aetiological role in some limb reduction anomalies.

Prevention of spinal neural tube defects in the curly tail mouse mutant by a specific effect of retinoic acid.

Curly tail mouse mutant embryos (ct/ct) develop spinal neural tube defects (NTD) in 54% of cases, comprising isolated tail flexion defects and spinal bifida with tail flexion defects. Both types of spinal NTD result from delayed closure of the posterior neuropore (PNP). Previous studies (Seller et al. [1979] Proc. R. Soc. Lond. Biol. 206:95-107; Seller and Perkins [1982] Prenat. Diagn. 2:297-300) described a paradoxical effect of retinoic acid (RA) on the phenotypic expression of the ct mutation: Treatment with low doses of RA on day 8 of gestation increased the incidence of total NTD, whereas low doses of RA administered on day 9 resulted in reduced incidence of total NTD. In order to investigate further the reported preventive effect of RA, we have carried out detailed analyses of the effects of maternal treatment with 5 mg/kg RA on the incidence of NTD at different developmental stages, and on the development and growth of ct/ct embryos. We found that 5 mg/kg RA reduces the incidence of spinal NTD in a stage-specific manner, without increasing the incidence of cranial NTD. The effect of RA is specific: There were no other alterations in morphogenesis, growth, development, resorption rate, or litter size. RA was more effective in the prevention of isolated tail flexion defects than of spina bifida. Prevention of isolated tail flexion defects was maximal (50% reduction) when RA was administered between 10 days 4 hours and 10 days 8 hours post coitum (p.c.) inclusive (24 to 34 somite stage).(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetic behaviour of the cranial neural epithelium during neurulation in the rat.

The kinetic behaviour of the cranial neuroepithelial cells of rat embryos during neurulation is described. Serial transverse sections of 4-, 8-, 12- and 16-somite-stage embryos show that differential mitosis does not play a part in the mechanisms responsible for effecting cranial neural tube closure. A constant cell number is found in the midbrain/hindbrain neural epithelium during all four stages; the mitotic spindle axes are oriented parallel to the long axis of the embryo, so that increase in cell number occurs in this direction only. Growth is only expressed by an expansion in the volume of the forebrain, which projects rostral to the notochordal tip. [3H]thymidine studies (using an in vitro culture technique) show no significant variation in the cell cycle time between the forebrain and the midbrain/anterior hindbrain neural epithelium. It is suggested that the neural epithelium is a fluid structure whose overall shape is strictly controlled while the cells within it flow towards and into the rapidly expanding forebrain.