Conditional inactivation of Fgf4 reveals complexity of signalling during limb bud development

Development of the vertebrate limb bud depends on reciprocal interactions between the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). Sonic hedgehog (SHH) and fibroblast growth factors (FGFs) are key signalling molecules produced in the ZPA and AER, respectively. Experiments in chicks suggested that SHH expression in the ZPA is maintained by FGF4 expression in the AER, and vice versa, providing a molecular mechanism for coordinating the activities of these two signalling centres. This SHH/FGF4 feedback loop model is supported by genetic evidence showing that Fgf4 expression is not maintained in Shh-/- mouse limbs. We report here that Shh expression is maintained and limb formation is normal when Fgf4 is inactivated in mouse limbs, thus contradicting the model. We also found that maintenance of Fgf9 and Fgf17 expression is dependent on Shh, whereas Fgf8 expression is not. We discuss a model in which no individual Fgf expressed in the AER (AER-Fgf) is solely necessary to maintain Shh expression, but, instead, the combined activities of two or more AER-Fgfs function in a positive feedback loop with Shh to control limb development.     

Fgf8 signalling from the AER is essential for normal limb development

Vertebrate limb development depends on signals from the apical ectodermal ridge (AER), which rims the distal tip of the limb bud. Removal of the AER in chick results in limbs lacking distal skeletal elements. Fibroblast growth factor (FGF) proteins can substitute for the AER (refs 4-7), suggesting that FGF signalling mediates AER activity. Of the four mouse Fgf genes (Fgf4 , Fgf8, Fgf9, Fgf17) known to display AER-specific expression domains within the limb bud (AER-Fgfs), only Fgf8 is expressed throughout the AER. Moreover, Fgf8 expression precedes that of other AER-Fgfs (refs 8-13), suggesting that Fgf8 may perform unique functions early in limb development. In mice, loss of function of Fgf4 (refs 13,14), Fgf9 (D. Ornitz, pers. comm.) or Fgf17 (ref. 15) has no effect on limb formation. We report here that inactivating Fgf8 in early limb ectoderm causes a substantial reduction in limb-bud size, a delay in Shh expression, misregulation of Fgf4 expression, and hypoplasia or aplasia of specific skeletal elements. Our data identify Fgf8 as the only known AER-Fgf individually necessary for normal limb development, and provide insight into the function of Fgf signalling from the AER in the normal outgrowth and patterning of the limb.     

A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant.

Early outgrowth of the vertebrate embryonic limb requires signalling by the apical ectodermal ridge (AER) to the progress zone (PZ), which in response proliferates and lays down the pattern of the presumptive limb in a proximal to distal progression. Signals from the PZ maintain the AER until the anlagen for the distal phalanges have been formed. The semidominant mouse mutant dactylaplasia (Dac) disrupts the maintenance of the AER, leading to truncation of distal structures of the developing footplate, or autopod. Adult Dac homozygotes thus lack hands and feet except for malformed single digits, whereas heterozygotes lack phalanges of the three middle digits. Dac resembles the human autosomal dominant split hand/foot malformation (SHFM) diseases. One of these, SHFM3, maps to chromosome 10q24 (Refs 6,7), which is syntenic to the Dac region on chromosome 19, and may disrupt the orthologue of Dac. We report here the positional cloning of Dac and show that it belongs to the F-box/WD40 gene family, which encodes adapters that target specific proteins for destruction by presenting them to the ubiquitination machinery. In conjuction with recent biochemical studies, this report demonstrates the importance of this gene family in vertebrate embryonic development.     

Vitamin A controls epithelial/mesenchymal interactions through Ret expression

Mutations or rearrangements in the gene encoding the receptor tyrosine kinase RET result in Hirschsprung disease, cancer and renal malformations. The standard model of renal development involves reciprocal signaling between the ureteric bud epithelium, inducing metanephric mesenchyme to differentiate into nephrons, and metanephric mesenchyme, inducing the ureteric bud to grow and branch. RET and GDNF (a RET ligand) are essential mediators of these epithelial-mesenchymal interactions. Vitamin A deficiency has been associated with widespread embryonic abnormalities, including renal malformations. The vitamin A signal is transduced by nuclear retinoic acid receptors (RARs). We previously showed that two RAR genes, Rara and Rarb2, were colocalized in stromal mesenchyme, a third renal cell type, where their deletion led to altered stromal cell patterning, impaired ureteric bud growth and downregulation of Ret in the ureteric bud. Here we demonstrate that forced expression of Ret in mice deficient for both Rara and Rarb2 (Rara(-/-)Rarb2(-/-)) genetically rescues renal development, restoring ureteric bud growth and stromal cell patterning. Our studies indicate the presence of a new reciprocal signaling loop between the ureteric bud epithelium and the stromal mesenchyme, dependent on Ret and vitamin A. In the first part of the loop, vitamin-A-dependent signals secreted by stromal cells control Ret expression in the ureteric bud. In the second part of the loop, ureteric bud signals dependent on Ret control stromal cell patterning.     

Early specification of limb muscle precursor cells by the homeobox gene Lbx1h.

During vertebrate embryogenesis, myogenic precursor cells of limb muscles delaminate from the ventro-lateral edge of the somitic dermomyotome and migrate to the limb buds, where they congregate into dorsal and ventral muscle masses. It has been proposed that the surrounding connective tissue controls muscle pattern formation in limbs. Regulatory molecules such as receptor tyrosine kinases like c-Met ( ref. 6) and those encoded by homeobox-containing genes, including c-Met (ref. 6), Tbx1 (ref. 7), Mox2 (ref. 8), Six1 and Six2 (ref. 9), Pitx2, Pax3 (refs 10,11) and Lbx1h (refs 12,13), are expressed in migrating limb precursor cells. The role of these genes in the patterning of limb muscles is unknown, although mutation of Pax3 or Met causes disruption of limb muscle development at an initial step, disturbing the epithelial-to-mesenchymal transition of the somitic epithelium. No limb muscle cells form in these mutants, and the early loss of myogenic precursor cells prevented an analysis of later functions of these genes during limb muscle development. Based on quail-chick chimaera studies, it was assumed that a cell-autonomous contribution of myogenic cells to the formation of individual limb muscles is negligible, and that an instructive role of limb mesenchyme is critical in this process. Here we show that Lbx1h determines migratory routes of muscle precursor cells in a cell-autonomous manner, thereby leading to the formation of distinct limb muscle patterns. Inactivation of Lbx1h, which is specifically expressed in migrating muscle precursor cells, led to a lack of extensor muscles in forelimbs and an absence of muscles in hindlimbs. The defect was caused by the failure of all muscle precursor cells of hindlimbs and of precursor cells of extensor muscles of forelimbs to migrate to their corresponding muscle anlagen. Our results demonstrate that Lbx1h is a key regulator of muscle precursor cell migration and is required for the acquisition of dorsal identities of forelimb muscles.     

Fgf8 is required for outgrowth and patterning of the limbs

The expression pattern and activity of fibroblast growth factor-8 (FGF8) in experimental assays indicate that it has important roles in limb development, but early embryonic lethality resulting from mutation of Fgf8 in the germ line of mice has prevented direct assessment of these roles. Here we report that conditional disruption of Fgf8 in the forelimb of developing mice bypasses embryonic lethality and reveals a requirement for Fgf8 in the formation of the stylopod, anterior zeugopod and autopod. Lack of Fgf8 in the apical ectodermal ridge (AER) alters expression of other Fgf genes, Shh and Bmp2.     

Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B

Inherited limb malformations provide a valuable resource for the identification of genes involved in limb development. Brachydactyly type B (BDB), an autosomal dominant disorder, is the most severe of the brachydactylies and characterized by terminal deficiency of the fingers and toes. In the typical form of BDB, the thumbs and big toes are spared, sometimes with broadening or partial duplication. The BDB1 locus was previously mapped to chromosome 9q22 within an interval of 7.5 cM (refs 9,10). Here we describe mutations in ROR2, which encodes the orphan receptor tyrosine kinase ROR2 (ref. 11), in three unrelated families with BDB1. We identified distinct heterozygous mutations (2 nonsense, 1 frameshift) within a 7-amino-acid segment of the 943-amino-acid protein, all of which predict truncation of the intracellular portion of the protein immediately after the tyrosine kinase domain. The localized nature of these mutations suggests that they confer a specific gain of function. We obtained further evidence for this by demonstrating that two patients heterozygous for 9q22 deletions including ROR2 do not exhibit BDB. Expression of the mouse mouse orthologue, Ror2, early in limb development indicates that BDB arises as a primary defect of skeletal patterning.     

The SH2 tyrosine phosphatase shp2 is required for mammalian limb development

The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis.     

No evidence of clonal somatic genetic alterations in cancer-associated fibroblasts from human breast and ovarian carcinomas

There is increasing evidence showing that the stromal cells surrounding cancer epithelial cells, rather than being passive bystanders, might have a role in modifying tumor outgrowth. The molecular basis of this aspect of carcinoma etiology is controversial. Some studies have reported a high frequency of genetic aberrations in carcinoma-associated fibroblasts (CAFs), whereas other studies have reported very low or zero mutation rates. Resolution of this contentious area is of critical importance in terms of understanding both the basic biology of cancer as well as the potential clinical implications of CAF somatic alterations. We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis of CAFs derived from breast and ovarian carcinomas using a 500K SNP array platform. Our data show conclusively that LOH and copy number alterations are extremely rare in CAFs and cannot be the basis of the carcinoma-promoting phenotypes of breast and ovarian CAFs.     

NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS) is a multiple malformation disorder characterized by dysmorphic facial features, mental retardation, growth delay and limb reduction defects. We indentified and characterized a new gene, NIPBL, that is mutated in individuals with CdLS and determined its structure and the structures of mouse, rat and zebrafish homologs. We named its protein product delangin. Vertebrate delangins have substantial homology to orthologs in flies, worms, plants and fungi, including Scc2-type sister chromatid cohesion proteins, and D. melanogaster Nipped-B. We propose that perturbed delangin function may inappropriately activate DLX genes, thereby contributing to the proximodistal limb patterning defects in CdLS. Genome analyses typically identify individual delangin or Nipped-B-like orthologs in diploid animal and plant genomes. The evolution of an ancestral sister chromatid cohesion protein to acquire an additional role in developmental gene regulation suggests that there are parallels between CdLS and Roberts syndrome.     

Wnt-beta-catenin signaling initiates taste papilla development

Fungiform taste papillae form a regular array on the dorsal tongue. Taste buds arise from papilla epithelium and, unusually for epithelial derivatives, synapse with neurons, release neurotransmitters and generate receptor and action potentials. Despite the importance of taste as one of our five senses, genetic analyses of taste papilla and bud development are lacking. We demonstrate that Wnt-beta-catenin signaling is activated in developing fungiform placodes and taste bud cells. A dominant stabilizing mutation of epithelial beta-catenin causes massive overproduction of enlarged fungiform papillae and taste buds. Likewise, genetic deletion of epithelial beta-catenin or inhibition of Wnt-beta-catenin signaling by ectopic dickkopf1 (Dkk1) blocks initiation of fungiform papilla morphogenesis. Ectopic papillae are innervated in the stabilizing beta-catenin mutant, whereas ectopic Dkk1 causes absence of lingual epithelial innervation. Thus, Wnt-beta-catenin signaling is critical for fungiform papilla and taste bud development. Altered regulation of this pathway may underlie evolutionary changes in taste papilla patterning.     

Ras-MAPK signaling promotes trophectoderm formation from embryonic stem cells and mouse embryos

In blastocyst chimeras, embryonic stem (ES) cells contribute to embryonic tissues but not extraembryonic trophectoderm. Conditional activation of HRas1(Q61L) in ES cells in vitro induces the trophectoderm marker Cdx2 and enables derivation of trophoblast stem (TS) cell lines that, when injected into blastocysts, chimerize placental tissues. Erk2, the downstream effector of Ras-mitogen-activated protein kinase (MAPK) signaling, is asymmetrically expressed in the apical membranes of the 8-cell-stage embryo just before morula compaction. Inhibition of MAPK signaling in cultured mouse embryos compromises Cdx2 expression, delays blastocyst development and reduces trophectoderm outgrowth from embryo explants. These data show that ectopic Ras activation can divert ES cells toward extraembryonic trophoblastic fates and implicate Ras-MAPK signaling in promoting trophectoderm formation from mouse embryos.     

Oral-facial-digital type I protein is required for primary cilia formation and left-right axis specification

The oral-facial-digital type I (OFD1) syndrome (OMIM 311200) is a human developmental disorder; affected individuals have craniofacial and digital abnormalities and, in 15% of cases, polycystic kidney. The disease is inherited as an X-linked dominant male-lethal trait. Using a Cre-loxP system, we generated knockout animals lacking Ofd1 and reproduced the main features of the disease, albeit with increased severity, possibly owing to differences of X inactivation patterns between human and mouse. We found failure of left-right axis specification in mutant male embryos, and ultrastructural analysis showed a lack of cilia in the embryonic node. Formation of cilia was defective in cystic kidneys from heterozygous females, implicating ciliogenesis as a mechanism underlying cyst development. In addition, we found impaired patterning of the neural tube and altered expression of the 5' Hoxa and Hoxd genes in the limb buds of mice lacking Ofd1, suggesting that Ofd1 could have a role beyond primary cilium organization and assembly.     

Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret

Almost 1% of human infants are born with urogenital abnormalities, many of which are linked to irregular connections between the distal ureters and the bladder. During development, ureters migrate by an unknown mechanism from their initial integration site in the Wolffian ducts up to the base of the bladder in a process that we call ureter maturation. Rara(-/-) Rarb2(-/-) mice display impaired vitamin A signaling and develop syndromic urogenital malformations similar to those that occur in humans, including renal hypoplasia, hydronephrosis and mega-ureter, abnormalities also seen in mice with mutations in the proto-oncogene Ret. Here we show that ureter maturation depends on formation of the 'trigonal wedge', a newly identified epithelial outgrowth from the base of the Wolffian ducts, and that the distal ureter abnormalities seen in Rara(-/-) Rarb2(-/-) and Ret(-/-) mutant mice are probably caused by a failure of this process. Our studies indicate that formation of the trigonal wedge may be essential for correct insertion of the distal ureters into the bladder, and that these events are mediated by the vitamin A and Ret signaling pathways.     

nagie oko, encoding a MAGUK-family protein, is essential for cellular patterning of the retina

A layered organization of cells is a common architectural feature of many neuronal formations. Mutations of the zebrafish gene nagie oko (nok) produce a severe disruption of retinal architecture, indicating a key role for this locus in neuronal patterning. We show that nok encodes a membrane-associated guanylate kinase-family scaffolding protein. Nok localizes to the vicinity of junctional complexes in retinal neuroepithelium and in the photoreceptor cell layer. Mosaic analysis indicates that the nok retinal patterning phenotype is not cell-autonomous. We propose that nok function in patterning of postmitotic neurons is mediated through neuroepithelial cells and is necessary for guiding neurons to their proper destinations in retinal laminae.     

Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis

Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.