John F. Fallon

Hedgehog-Regulated Processing of Gli3 Produces an Anterior/Posterior Repressor Gradient in the Developing Vertebrate Limb

Ci/Gli zinc finger proteins mediate the transcriptional effects of Hedgehog protein signals. In Drosophila, Ci action as transcriptional repressor or activator is contingent upon Hedgehog-regulated, PKA-dependent proteolytic processing. We demonstrate that PKA-dependent processing of vertebrate Gli3 in developing limb similarly generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. The resulting anterior/posterior Gli3 repressor gradient can be perturbed by mutations of Gli3 in human genetic syndromes or by misregulation of Gli3 processing in the chicken mutant talpid2, producing a range of limb patterning malformations. The high relative abundance and potency of Gli3 repressor suggest specialization of Gli3 and its products for negative Hedgehog pathway regulation.

Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity

Most current models propose Sonic hedgehog (Shh) as the primary determinant of anteroposterior development of amniote limbs. Shh protein is said to be required to direct the formation of skeletal elements and to specify digit identity through dose-dependent activation of target gene expression. However, the identity of genes targeted by Shh, and the regulatory mechanisms controlling their expression, remain poorly understood. Gli3 (the gene implicated in human Greig cephalopolysyndactyly syndrome) is proposed to negatively regulate Shh by restricting its expression and influence to the posterior mesoderm. Here we report genetic analyses in mice showing that Shh and Gli3 are dispensable for formation of limb skeletal elements: Shh-/- Gli3-/- limbs are distally complete and polydactylous, but completely lack wild-type digit identities. We show that the effects of Shh signalling on skeletal patterning and ridge maintenance are necessarily mediated through Gli3. We propose that the function of Shh and Gli3 in limb skeletal patterning is limited to refining autopodial morphology, imposing pentadactyl constraint on the limbs polydactyl potential, and organizing digit identity specification, by regulating the relative balance of Gli3 transcriptional activator and repressor activities.

Fibroblast growth factors as multifunctional signaling factors.

The fibroblast growth factor (FGF) family consists of at least 15 structurally related polypeptide growth factors. Their expression is controlled at the levels of transcription, mRNA stability, and translation. The bioavailability of FGFs is further modulated by posttranslational processing and regulated protein trafficking. FGFs bind to receptor tyrosine kinases (FGFRs), heparan sulfate proteoglycans (HSPG), and a cysteine-rich FGF receptor (CFR). FGFRs are required for most biological activities of FGFs. HSPGs alter FGF-FGFR interactions and CFR participates in FGF intracellular transport. FGF signaling pathways are intricate and are intertwined with insulin-like growth factor, transforming growth factor-beta, bone morphogenetic protein, and vertebrate homologs of Drosophila wingless activated pathways. FGFs are major regulators of embryonic development: They influence the formation of the primary body axis, neural axis, limbs, and other structures. The activities of FGFs depend on their coordination of fundamental cellular functions, such as survival, replication, differentiation, adhesion, and motility, through effects on gene expression and the cytoskeleton.

Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage

BACKGROUND Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH. RESULTS We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud. CONCLUSIONS Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

Unique SMAD1/5/8 activity at the phalanx-forming region determines digit identity

The zone of polarizing activity is the primary signaling center controlling anterior–posterior patterning of the amniote limb bud. The autopodial interdigits (IDs) are secondary signaling centers proposed to determine digit identity by acting on the cells of the digital ray. Here, we focus on events accompanying digital fate determination and define a region of the digital ray that expresses Sox9 and Bmpr1b and is phosphorylated-SMAD1/5/8 (p-SMAD1/5/8) positive. We name this region the phalanx-forming region (PFR), and show that the PFR cells arise from the distal subridge mesenchyme of digital ray. This phalanx-forming cell lineage is subsequently committed to the cartilage lineage; the fate of these cells is initially labile but becomes fixed as they are incorporated into the condensed cartilage of the digit primordium. Using an in vivo reporter assay, we establish that each digital PFR has a unique p-SMAD1/5/8 activity signature. In addition, we show that changes in this activity correlate with the identity of the digit that forms after experimental manipulation, supporting the idea that threshold signaling levels can lead to different developmental outcomes in a morphogenetic field. Our data define the molecular profile of the PFR, and we propose a model for understanding formation and variation of digits during autopodial development.

The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb.

We have analyzed a new limb mutant in the chicken that we name oligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod (ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that the ozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. Neither Ptc1 nor Gli1 are detectable in mutant limb buds. However, Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation of Hoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II, and expression is more severely affected in the more 5′ genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern to ozd limbs; however, retinoic acid fails to induce Shh in ozd limb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh-/- mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.

Spatial and temporal patterns of cell death in limb bud mesoderm after apical ectodermal ridge removal

Abstract A spatiotemporal pattern of cell death occurred in the chick wing and leg bud mesoderm after removal of apical ectodermal ridge at stages 18–20. Cells died in a region extending from the limb bud distal surface to 150–200 μm into the mesoderm. Limb buds from which ridge was removed at later stages in development did not exhibit a spatiotemporal pattern of cell death. In control experiments in which dorsal ectoderm was removed, a pattern of cell death did not occur. Removal of the ridge and part of the 150- to 200-μm zone of prospective cell death resulted in cell death in an area approximately equal to the amount of the zone remaining. After removal of all of the prospective zone of cell death plus the apical ridge, cell death was observed in the remaining limb bud mesoderm. In these limb buds, cell death occurred in a region in which it had not been seen in limb bud with apical ridge alone removed. We conclude that at stages 18–20 the mesodermal cells 150–200 μm beneath the ridge require the apical ridge to survive. More proximal mesodermal cells do not die after ridge removal alone, but apparently require the presence of the more distal mesoderm to survive. Whether this is a requirement for something intrinsic to the distal mesoderm or something it possesses by way of the ridge is unknown. After stage 23, the limb mesoderm cells do not die when the apical ridge is removed. Nevertheless, at the later stages, ridge continues to be required for limb bud proximal-distal elongation and the differentiation of distal limb elements.

The Development of Archosaurian First-Generation Teeth in a Chicken Mutant

Modern birds do not have teeth. Rather, they develop a specialized keratinized structure, called the rhamphotheca, that covers the mandible, maxillae, and premaxillae. Although recombination studies have shown that the avian epidermis can respond to tooth-inductive cues from mouse or lizard oral mesenchyme and participate in tooth formation, attempts to initiate tooth development de novo in birds have failed. Here, we describe the formation of teeth in the talpid2 chicken mutant, including the developmental processes and early molecular changes associated with the formation of teeth. Additionally, we show recapitulation of the early events seen in talpid2 after in vivo activation of beta-catenin in wild-type embryos. We compare the formation of teeth in the talpid2 mutant with that in the alligator and show the formation of decidedly archosaurian (crocodilian) first-generation teeth in an avian embryo. The formation of teeth in the mutant is coupled with alterations in the specification of the oral/aboral boundary of the jaw. We propose an epigenetic model of the developmental modification of dentition in avian evolution; in this model, changes in the relative position of a lateral signaling center over competent odontogenic mesenchyme led to loss of teeth in avians while maintaining tooth developmental potential.

Hind Limb Malformations in Free-Living Northern Leopard Frogs (Rana pipiens) From Maine, Minnesota, and Vermont Suggest Multiple Etiologies

BACKGROUND Reports of malformed frogs have increased throughout the North American continent in recent years. Most of the observed malformations have involved the hind limbs. The goal of this study was to accurately characterize the hind limb malformations in wild frogs as an important step toward understanding the possible etiologies. METHODS During 1997 and 1998, 182 recently metamorphosed northern leopard frogs (Rana pipiens) were collected from Minnesota, Vermont, and Maine. Malformed hind limbs were present in 157 (86%) of these frogs, which underwent necropsy and radiographic evaluation at the National Wildlife Health Center. These malformations are described in detail and classified into four major categories: (1) no limb (amelia); (2) multiple limbs or limb elements (polymelia, polydactyly, polyphalangy); (3) reduced limb segments or elements (phocomelia, ectromelia, ectrodactyly, and brachydactyly; and (4) distally complete but malformed limb (bone rotations, bridging, skin webbing, and micromelia). RESULTS Amelia and reduced segments and/or elements were the most common finding. Frogs with bilateral hind limb malformations were not common, and in only eight of these 22 frogs were the malformations symmetrical. Malformations of a given type tended to occur in frogs collected from the same site, but the types of malformations varied widely among all three states, and between study sites within Minnesota. CONCLUSIONS Clustering of malformation type suggests that developmental events may produce a variety of phenotypes depending on the timing, sequence, and severity of the environmental insult. Hind limb malformations in free-living frogs transcend current mechanistic explanations of tetrapod limb development.

Single base pair change in the long‐range Sonic hedgehog limb‐specific enhancer is a genetic basis for preaxial polydactyly

In most instances of preaxial polydactyly (PPD), Sonic Hedgehog (Shh), an essential limb patterning signal, is ectopically expressed in an anterior region of the developing limb in addition to the normal posterior domain. It is thought that this anterior Shh expression leads directly to the development of the extra digits. Recent reports have identified a conserved limb‐specific Shh enhancer ∼1 megabase upstream of the Shh transcription initiation site, and individual base pair changes within this region are associated with PPD. We report here that a single base pair change within this enhancer is sufficient to drive β‐galactosidase expression in both anterior and posterior limb domains, similar to Shh expression in animal PPD models, whereas a wild‐type construct is expressed only in the posterior limb, similar to Shh expression in normal embryos. These findings provide the first direct evidence that a single base pair change within the limb‐specific Shh enhancer acts as a genetic basis for PPD. Developmental Dynamics 232:345–348, 2005.