Cheryll Tickle

FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb

The apical ectodermal ridge plays a key role in limb development. We show that recombinant FGF-4 can substitute for the ridge to provide all the signals necessary for virtually complete outgrowth and patterning of the chick limb. FGF-4 stimulates proliferation of cells in the distal mesenchyme and maintains a signal from the posterior to the distal mesenchyme that appears to be required for elaboration of skeletal elements in the normal proximodistal sequence. Moreover, retinoic acid, which is capable of providing polarizing activity, can supply this signal. This suggests that polarizing activity plays a role in patterning along the proximodistal axis, in addition to its well-established role in anteroposterior patterning. Taken together, the data suggest a simple mechanism whereby FGF-4 links growth and pattern formation during limb development.

Fibroblast growth factors induce additional limb development from the flank of chick embryos

Fibroblast growth factors (FGFs) act as signals in the developing limb and can maintain proliferation of limb bud mesenchyme cells. Remarkably, beads soaked in FGF-1, FGF-2, or FGF-4 and placed in the presumptive flank of chick embryos induce formation of ectopic limb buds, which can develop into complete limbs. The entire flank can produce additional limbs, but generally wings are formed anteriorly and legs posteriorly. FGF application activates Sonic hedgehog in cells with polarizing potential to make a discrete polarizing region. Hoxd-13 is also expressed in the ectopic bud, and an apical ectodermal ridge forms. A limb bud is thus established that can generate the appropriate signals to develop into a complete limb. The additional limbs have reversed polarity. This can be explained by the distribution of cells in the flank with potential polarizing activity. The results suggest that local production of an FGF may initiate limb development.

Developmental basis of limblessness and axial patterning in snakes

The evolution of snakes involved major changes in vertebrate body plan organization, but the developmental basis of those changes is unknown. The python axial skeleton consists of hundreds of similar vertebrae, forelimbs are absent and hindlimbs are severely reduced. Combined limb loss and trunk elongation is found in many vertebrate taxa, suggesting that these changes may be linked by a common developmental mechanism. Here we show that Hox gene expression domains are expanded along the body axis in python embryos, and that this can account for both the absence of forelimbs and the expansion of thoracic identity in the axial skeleton. Hindlimb buds are initiated, but apical-ridge and polarizing-region signalling pathways that are normally required for limb development are not activated. Leg bud outgrowth and signalling by Sonic hedgehog in pythons can be rescued by application of fibroblast growth factor or by recombination with chick apical ridge. The failure to activate these signalling pathways during normal python development may also stem from changes in Hox gene expression that occurred early in snake evolution.

A Comprehensive Collection of Chicken cDNAs

Birds have played a central role in many biological disciplines, particularly ecology, evolution, and behavior. The chicken, as a model vertebrate, also represents an important experimental system for developmental biologists, immunologists, cell biologists, and geneticists. However, genomic resources for the chicken have lagged behind those for other model organisms, with only 1845 nonredundant full-length chicken cDNA sequences currently deposited in the EMBL databank. We describe a large-scale expressed-sequence-tag (EST) project aimed at gene discovery in chickens (http://www.chick.umist.ac.uk). In total, 339,314 ESTs have been sequenced from 64 cDNA libraries generated from 21 different embryonic and adult tissues. These were clustered and assembled into 85,486 contiguous sequences (contigs). We find that a minimum of 38% of the contigs have orthologs in other organisms and define an upper limit of 13,000 new chicken genes. The remaining contigs may include novel avian specific or rapidly evolving genes. Comparison of the contigs with known chicken genes and orthologs indicates that 30% include cDNAs that contain the start codon and 20% of the contigs represent full-length cDNA sequences. Using this dataset, we estimate that chickens have approximately 35,000 genes in total, suggesting that this number may be a characteristic feature of vertebrates.

A quantitative analysis of the effect of all-trans-retinoic acid on the pattern of chick wing development

Small, positively charged beads that slowly release known amounts of all-trans-retinoic acid have been implanted below the apical ectodermal ridge at the anterior margin (opposite somite 16) of wing buds of 3 1/2 day-old chick embryos. The continuous release of retinoic acid is shown to create an anteroposterior concentration gradient of retinoic acid in the limb field that is stable with time, despite the fact that this compound is metabolized by the limb tissue. With beads that release increasing amounts of retinoic acid, the normal 234 digit pattern is progressively altered to a 2234, to a 32234, and then to a 432234 pattern. The tissue concentrations of all-trans-retinoic acid required to change the digit pattern in this way range between 1 and 25 nM. When the same amounts of retinoic acid are released from posteriorly implanted beads (placed below the apical ectodermal ridge opposite somite border 19/20 or somite 20), the normal digit pattern is unaffected. Implantations of beads that release all-trans-retinoic acid are thus identical in their effect to grafts of cells from the limb polarizing region, which cause similar dose-dependent changes in the digit pattern when grafted to the anterior margin of the bud (but not when grafted opposite somites 19 or 20). Because of the low concentrations of retinoic acid required for its biological effect, the graded response observed, and the fact that a concentration gradient is established across the limb field, all-trans-retinoic acid closely mimics the putative morphogen that has been postulated to be emitted by polarizing region cells during normal development.

Patterning Systems—From One End of the Limb to the Other

A combination of embryology and gene identification has led us to the current view of vertebrate limb development, in which a series of three interlocking patterning systems operate sequentially over time. This review describes current understanding of these regulatory mechanisms and how they form a framework for future analysis of limb patterning.

Identification of Novel Roles of the Cytochrome P450 System in Early Embryogenesis: Effects on Vasculogenesis and Retinoic Acid Homeostasis

ABSTRACT The cytochrome P450-dependent monooxygenase system catalyzes the metabolism of xenobiotics and endogenous compounds, including hormones and retinoic acid. In order to establish the role of these enzymes in embryogenesis, we have inactivated the system through the deletion of the gene for the electron donor to all microsomal P450 proteins, cytochrome P450 reductase (Cpr). Mouse embryos homozygous for this deletion died in early to middle gestation (∼9.5 days postcoitum [dpc]) and exhibited a number of novel phenotypes, including the severe inhibition of vasculogenesis and hematopoiesis. In addition, defects in the brain, limbs, and cell types where CPR was shown to be expressed were observed. Some of the observed abnormalities have been associated with perturbations in retinoic acid homeostasis in later embryogenesis. Consistent with this possibility, embryos at 9.5 dpc had significantly elevated levels of retinoic acid and reduced levels of retinol. Further, some of the observed phenotypes could be either reversed or exacerbated by decreasing or increasing maternal retinoic acid exposure, respectively. Detailed analysis demonstrated a close relationship between the observed phenotype and the expression of genes controlling vasculogenesis. These data demonstrate that the cytochrome P450 system plays a key role in early embryonic development; this process appears to be, at least in part, controlled by regional concentrations of retinoic acid and has profound effects on blood vessel formation.

Integration of growth and specification in chick wing digit-patterning

In the classical model of chick wing digit-patterning, the polarizing region—a group of cells at the posterior margin of the early bud—produces a morphogen gradient, now known to be based on Sonic hedgehog (Shh), that progressively specifies anteroposterior positional identities in the posterior digit-forming region. Here we add an integral growth component to this model by showing that Shh-dependent proliferation of prospective digit progenitor cells is essential for specifying the complete pattern of digits across the anteroposterior axis. Inhibiting Shh signalling in early wing buds reduced anteroposterior expansion, and posterior digits were lost because all prospective digit precursors formed anterior structures. Inhibiting proliferation also irreversibly reduced anteroposterior expansion, but instead anterior digits were lost because all prospective digit precursors formed posterior structures. When proliferation recovered in such wings, Shh transcription was maintained for longer than normal, suggesting that duration of Shh expression is controlled by a mechanism that measures proliferation. Rescue experiments confirmed that Shh-dependent proliferation controls digit number during a discrete time-window in which Shh-dependent specification normally occurs. Our findings that Shh signalling has dual functions that can be temporally uncoupled have implications for understanding congenital and evolutionary digit reductions.

Negative Feedback Regulation of FGF Signaling Levels by Pyst1/MKP3 in Chick Embryos

BACKGROUND The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. RESULTS We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. CONCLUSIONS Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.

The chicken as a model for large-scale analysis of vertebrate gene function.

The chicken has been an important experimental system for developmental biology, immunology and microbiology, having led to many fundamental discoveries. The increase in genomic resources, easy access to the embryo and the application of RNA interference mean that it will be easy and quick to use chick embryos to screen the function of many genes during embryonic development. So, it seems likely that the chicken will increasingly be the system of choice for many vertebrate biologists who are interested in gene function.