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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".

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.

The mouse doublefoot (Dbf) mutant exhibits preaxial polydactyly in association with craniofacial defects. This mutation has previously been mapped to mouse chromosome 1. We have used a positional cloning strategy, coupled with a comparative sequencing approach using available human draft sequence, to identify putative candidates for the Dbf gene in the mouse and in homologous human region. We have constructed a high-resolution genetic map of the region, localizing the mutation to a 0.4-cM (+/-0.0061) interval on mouse chromosome 1. Furthermore, we have constructed contiguous BAC/PAC clone maps across the mouse and human Dbf region. Using existing markers and additional sequence tagged sites, which we have generated, we have anchored the physical map to the genetic map. Through the comparative sequencing of these clones we have identified 35 genes within this interval, indicating that the region is gene-rich. From this we have identified several genes that are known to be differentially expressed in the developing mid-gestation mouse embryo, some in the developing embryonic limb buds. These genes include those encoding known developmental signaling molecules such as WNT proteins and IHH, and we provide evidence that these genes are candidates for the Dbf mutation.

This review describes the evolutionary history of the mammalian skull vault as a basis for understanding its complex structure. Current information on the developmental tissue origins of the skull vault bones (mesoderm and neural crest) is assessed for mammals and other tetrapods. This information is discussed in the context of evolutionary changes in the proportions of the skull vault bones at the sarcopterygian-tetrapod transition. The dual tissue origin of the skull vault is considered in relation to the molecular mechanisms underlying osteogenic cell proliferation and differentiation in the sutural growth centres and in the proportionate contributions of different sutures to skull growth.

The head is anatomically the most sophisticated part of the body and its evolution was fundamental to the origin of vertebrates; understanding its development is a formidable problem in biology. A synthesis of embryology, evolution and mouse genetics is shaping our understanding of head development and in this review we discuss its application to studies of human craniofacial malformations. Many of these disorders have their origins in specific embryological processes, including abnormalities of brain patterning, of the migration and fusion of tissues in the face, and of bone differentiation in the skull vault.

Retinoic acid, when administered to pregnant mice on d 11.0 of gestation, causes limb skeletal abnormalities consisting of reduced digital number, shortening of the long bones and delayed ossification. We show here that these effects are correlated with a decrease in cell proliferation within 5 h of retinoic acid administration, specifically in the posterior half of the distal limb bud mesenchyme, from which the distal skeletal elements are generated. There is a specific downregulation of Fgf4, a gene known to be involved in limb bud outgrowth and expressed only in the posterior part of the apical ectodermal ridge; Fgf8, which is expressed throughout the apical ectodermal ridge, is unaffected. The reduction in Fgf4 expression is not accompanied by downregulation of Shh, nor of its receptor and downstream target gene Ptc, suggesting that the skeletal reduction defects induced by retinoic acid are mediated specifically by FGF4-induced skeletogenic mesenchymal cell proliferation.

Activating mutations of genes encoding the transmembrane tyrosine kinase receptors fibroblast growth factor receptors (FGFRs)1-3, and haploinsufficiency of the transcription factor TWIST, cause human craniosynostosis syndromes that typically involve the coronal suture. We have investigated the functional roles of these genes in development of the coronal suture in mouse fetuses, and tested the effects of increasing FGFR signalling by applying exogenous FGF2 to the suture. The results indicate that the proliferation-differentiation balance in normal sutural development involves a gradient of extracellular FGF from the region of differentiation, in which Fgfr1 is expressed, to the sutural mesenchyme, in which low levels of FGF are associated with Fgfr2 expression in osteogenic stem cells. Experimental increase of sutural FGF levels leads to down-regulation of Fgfr2, up-regulation of Fgfr1, up-regulation of the osteogenic differentiation gene Osteopontin, and cessation of proliferation. Twist is expressed in the midsutural mesenchyme and is partially co-expressed with Fgfr2, consistent with the possibility that it is involved in maintaining proliferation through regulating transcription of Fgfr2.

Inherited defects of skull ossification often manifest as symmetric parietal foramina (PFM; MIM 168500). We previously identified mutations of MSX2 in non-syndromic PFM and demonstrated genetic heterogeneity. Deletions of 11p11-p12 (proximal 11p deletion syndrome, P11pDS; MIM 601224) are characterized by multiple exostoses, attributable to haploinsufficiency of EXT2 and PFM. Here we identify ALX4, which encodes a paired-related homeodomain transcription factor, as the PFM disease gene in P11pDS.

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.

All vertebrate embryos require retinoic acid (RA) for fulfilment of the developmental program encoded in the genome. In mammals, maternal homeostatic mechanisms minimize variation of retinoid levels reaching the embryo. Retinol is transported as a complex with retinol-binding protein (RBP): transplacental transfer of retinol and its uptake by the embryonic tissues involves binding to an RBP receptor at the cell surface. Embryonic tissues in which this receptor is present also contain the retinol-binding protein CRBP I and the enzymes involved in RA synthesis; the same tissues are particularly vulnerable to vitamin A deficiency. In the nucleus, the RA signal is transduced by binding to a heterodimeric pair of retinoid receptors (RAR/RXR). In general, the receptors show functional plasticity, disruption of one RAR or RXR gene having minor or no effects on embryogenesis. However, genetic studies indicate that RXR alpha is essential for normal development of the heart and eye. Excess RA causes abnormalities of many systems; altered susceptibility to RA excess in mice lacking RAR gamma or RXR alpha suggests that the teratogenic signal is transduced through different receptors compared with physiological RA function in the same tissue.

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.

We report the morphogenesis of a new mouse mutant, Doublefoot (Dbf). The major phenotypic features involve the limb and craniofacial regions. There is polydactyly of all 4 limbs, with typically 6-8 digits per limb. All of the digits are triphalangeal; some show bifurcations and some are not attached to the carpus/tarsus. The carpus and tarsus are broader than normal, and their elements are partially fused. There are also tibial defects. Mutant embryos show a diencephalic bulge on d 10.0, with older animals exhibiting broadened and bulbous skulls sometimes with an additional midline skeletal element, shortened snouts and bulging eyes. Homozygotes, which do not survive beyond d 15, show midline facial clefting. In this study of the embryonic and fetal development of Dbf animals, we focus on the morphogenesis of the limbs and head, and discuss the possible molecular developmental mechanisms.

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.

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.

Retinoic acid (RA) is essential for normal embryonic development. In mammals it is sequestered from the maternal circulation in the form of retinol. In rodents, embryonic uptake relies upon the presence of retinol binding protein (RBP) in the yolk sac, probably involving an RBP receptor. The molecular activity of RA in the nucleus is well established, but less is known about cytoplasmic events including tissue-specific intraembryonic RA synthesis and intracellular transport of both retinol and RA. The cellular binding proteins for retinol and RA may play important roles in these processes.

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.

The technique of whole embryo culture has made significant contributions to understanding the mechanisms of morphogenesis in mammalian embryos, especially with respect to cranial neurulation and neural crest cell migration. This study traces the fate of two specifically mammalian structures, the preotic and otic sulci. Their formation at the 1/2- and 3-somite stages respectively, divides the hindbrain neuroepithelium into prorhombomeres A, B and C. The preotic sulcus is a deeply recessed structure that forms the rostral boundary of expression of both Hoxb-2 and the first domain of Krox-20. The otic sulcus is a shallow concavity in which the second Krox-20 domain is expressed. DiI labeling followed by whole embryo culture confirmed that the later fate of the preotic sulcus is the rhombomere 2/3 boundary, and the fate of the otic sulcus is the cranial part of rhombomere 5. Structurally, the preotic and otic sulci show no specialization with respect to actin, tubulin or proteoglycans, but their maintenance depends on contact with the subjacent mesenchyme. Their formation is inhibited by exposure of embryos to retinoic acid prior to the onset of somitic segmentation, indicating that the molecular events governing prorhombomeric subdivision of the hindbrain are retinoic acid-sensitive. The preotic sulcus may be essential for neuroepithelial cell movement towards and into the rapidly enlarging forebrain; the otic sulcus may simply delineate the caudal boundary of prorhombomere B, an area with a discrete neural crest cell population discontinuous with those rostral and caudal to it. Understanding the positional relationships of the preotic and otic sulci to later rhombomeric segments makes them useful landmarks for experimental purposes, but there is no evidence that prorhombomeres are functionally significant as the precursors of rhombomeric segments.

AP-2 is a recent significant addition to the list of transcription factors that have been demonstrated by targeted gene disruption to be essential for normal development. Two recent reports of AP-2 null mutant mice indicate that AP-2 holds a key position in the network of genes and proteins controlling developmental pattern and morphogenesis, and that it is particularly important for development of the cranial region and for midline fusions.

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.

Apert syndrome is a distinctive human malformation characterized by craniosynostosis and severe syndactyly of the hands and feet. It is caused by specific missense substitutions involving adjacent amino acids (Ser252Trp or Pro253Arg) in the linker between the second and third extracellular immunoglobulin domains of fibroblast growth factor receptor 2 (FGFR2). We have developed a simple PCR assay for these mutations in genomic DNA, based on the creation of novel (SfiI) and (BstUI) restriction sites. Analysis of DNA from 70 unrelated patients with Apert syndrome showed that 45 had the Ser252Trp mutation and 25 had the Pro253Arg mutation. Phenotypic differences between these two groups of patients were investigated. Significant differences were found for severity of syndactyly and presence of cleft palate. The syndactyly was more severe with the Pro253Arg mutation, for both the hands and the feet. In contrast, cleft palate was significantly more common in the Ser252Trp patients. No convincing differences were found in the prevalence of other malformations associated with Apert syndrome. We conclude that, although the phenotype attributable to the two mutations is very similar, there are subtle differences. The opposite trends for severity of syndactyly and cleft palate in relation to the two mutations may relate to the varying patterns of temporal and tissue-specific expression of different fibroblast growth factors, the ligands for FGFR2.