Distal Limb Bud Research Articles

Rethinking the proximodistal axis of the vertebrate limb in the molecular era

The development of the vertebrate limb has long served as a paradigm for understanding the fundamental processes by which an undifferentiated field of cells gains spatial pattern and undergoes coordinated differentiation to produce the exquisitely complex structures that characterize our functional anatomy. The limb bud emerges from the flank as a mound of seemingly homogeneous mesenchymal cells within an ectodermal jacket. Yet within days this mass of cells gives rise to skeletal elements, muscles, and connective tissues organized in the form of a mature limb. Looking at one’s own arm or leg, this organization can immediately be appreciated along three different axes: The back of the hand (dorsal) is different from the palm (ventral), the thumb (anterior) is distinct from the little finger (posterior), and the upper arm (proximal) is different from the lower arm and the hand (distal). The process by which differences along the proximodistal axis are established has been particularly controversial, with competing models proposed to explain the outcome of both classical and genetic manipulations. However, if one examines the two major models that have been previously proposed, neither of them is tenable in the context of our current knowledge of gene activity in the developing limb. Proximodistal patterning therefore needs to be placed into a new framework based directly on the molecular data. The first key insight into the process of limb patterning came almost 60 yr ago when John Saunders discovered that the apical ectodermal ridge (AER), a thickened ridge of ectodermal cells that runs along the anterior– posterior axis of the distal limb bud (equivalent to a ridge running along the distal edge of the hand from which the finger tips will eventually form) is necessary for the successful outgrowth of the limb along the proximodistal axis (Saunders 1948). To explain how different structures arise at different proximodistal levels as the limb bud grows out, a model was subsequently proposed based on progressive specification of increasingly distal cell fates in a domain ∼300 µm deep directly below the AER (Summerbell et al. 1973). According to this view, under the influence of the AER, cells are maintained in a so-called progress zone. These cells continuously acquire ever more distal positional information through the influence of an internal clock that is kept active as long as the cells receive signaling from the overlying AER. This process gives the cells a distal fate proportional to the length of time the cells remain in the progress zone. When cells move out of the range of AER signaling— i.e., out of the progress zone—the clock stops and their proximodistal fate therefore becomes fixed. This happens continuously, as all the cells in the progress zone—indeed, all of the cells of the limb bud—are dividing. As more cells are produced, only the members of the population closest to

A CRITICAL ROLE FOR THE SHH‐FGF LOOP DURING THE INITIATION OF LIMB REGENERATION

Limb outgrowth is initiated and maintained by the secretion of FGFs in the apical ectodermal ridge (AER), and patterning regulated by SHH emanating from the posteriorly positioned zone of polarizing activity (ZPA). These factors are tightly linked during progressive limb formation by the SHH-FGF loop. Distal limb bud amputation (removing AER and ZPA) followed by application of FGF to the posterior (but not anterior) stump reactivates SHH expression and regeneration of the distal limb structures. We wondered whether SHH was sufficient to induce regenerative capacity and FGF-responsiveness in limb mesoderm. To determine the role of SHH in limb regeneration, we amputated developing wings (HH 23) and ectopically expressed SHH by confined microeclectroporation (CMEP) in the anterior and posterior stump margins in the presence or absence of FGF soaked beads. Also we ectopically expressed SHH in the anterior margin at HH 19 by CMEP, then applied FGF soaked beads to the anterior margin 24 h later (HH 23-24). Chic...

Novel molecular mechanisms regulating Shh expression and limb patterning

Normal patterning of the developing limb requires tight restriction of Sonic hedgehog (Shh) mRNA to the posterior margin of the limb. While several positive and negative regulatory factors have been shown to control Shh expression, these factors do not adequately explain the exquisite domain of Shh. Using several approaches, we provide novel insight into the molecular mechanisms which regulate Shh expression. First, we identify the dorsal-ventral ectoderm border proximal to the AER as a new signaling center in limb development that serves to regulate the restricted expression of Tbx2 and Shh. In separate experiments, we have analyzed the function of ETV4 and ETV5, two members of the ETS family of transcriptional factors, which we show are expressed in the distal mesenchyme of developing limb bud in a FGF dependent manner. Surprisingly, repressing ETV4/5 transcriptional activity in the limb mesenchyme resulted in polydactyly and ectopic induction of Shh expression in the anterior distal limb bud, indicating ETV4/5 normally function to suppress Shh expression in the anterior mesenchyme. Further, we demonstrate that ETV4/5 positively contribute to FGF-controlled limb growth. Taken together, our work expands the current understanding of molecular mechanisms regulating Shh expression and pattern formation in the limb.

Transgenic analysis of Hoxd gene regulation during digit development

In tetrapods, posterior Hoxd genes (from groups 10 to 13) are necessary to properly pattern the developing autopods, including the number and identities of digits. Their coordinated expression is achieved by sharing a global control region (GCR), which was isolated and localized 200 kb 5′ (centromeric) of the gene cluster. However, in transgenic assays, the GCR was unable to fully recapitulate all aspects of the endogenous Hoxd expression patterns during distal limb development. In this paper, we further analyze the regulatory potential of this locus and report the characterization of Prox, a second enhancer element that contributes to the transcriptional activity of posterior Hoxd genes in developing distal limb buds. We show that the GCR and Prox elements complement each other and work in combination to correctly establish the late phase of Hoxd genes expression. Based on DNA sequence conservation and transgenic assays, we discuss the functions of these regulatory regions as well as a potential evolutionary scheme accounting for their emergence along with the evolution of tetrapod limbs.

Specification of cell fate along the proximal-distal axis in the developing chick limb bud

Pattern formation along the proximal-distal (PD) axis in the developing limb bud serves as a good model for learning how cell fate and regionalization of domains, which are essential processes in morphogenesis during development, are specified by positional information. In the present study, detailed fate maps for the limb bud of the chick embryo were constructed in order to gain insights into how cell fate for future structures along the PD axis is specified and subdivided. Our fate map revealed that there is a large overlap between the prospective autopod and zeugopod in the distal limb bud at an early stage (stage 19), whereas a limb bud at this stage has already regionalized the proximal compartments for the prospective stylopod and zeugopod. A clearer boundary of cell fate specifying the prospective autopod and zeugopod could be seen at stage 23, but cell mixing was still detectable inside the prospective autopod region at this stage. Detailed analysis of HOXA11 AND HOXA13 expression at single cell resolution suggested that the cell mixing is not due to separation of some different cell populations existing in a mosaic. Our findings suggest that a mixable unregionalized cell population is maintained in the distal area of the limb bud, while the proximal region starts to be regionalized at the early stage of limb development.

Progressive mRNA decay establishes an mkp3 expression gradient in the chick limb bud

The apical ectodermal ridge (AER) controls limb outgrowth and patterning, such that its removal causes changes in mesodermal gene expression, cell death and limb truncation. Fibroblast growth factor (FGF) family members are expressed in the AER and can rescue limb bud outgrowth after AER removal. Cells localized underneath the AER are maintained in an undifferentiated state by the FGFs produced by the AER. MAPK phosphatase 3 ( mkp3) is a downstream effector of FGF8 signalling during limb bud development and is expressed in the distal limb mesenchyme. The present work evidences a gradient of mkp3 transcripts along the chick limb bud, in a distal to proximal direction. mkp3 transcription occurs only in the most distal limb bud cells and its mRNA gradient throughout the limb results from progressive mRNA decay. We show that FGF8-soaked beads induce ectopic mkp3 expression, indicating that AER-derived FGF8 protein may activate mkp3 in the distal mesenchyme.

Differential regulation of gene expression in the digit forming area of the mouse limb bud by SHH and gremlin 1/FGF-mediated epithelial-mesenchymal signalling

Spatially and temporally coordinated changes in gene expression are crucial to orderly progression of embryogenesis. We combine mouse genetics with experimental manipulation of signalling to analyze the kinetics by which the SHH morphogen and the BMP antagonist gremlin 1 (GREM1) control gene expression in the digit-forming mesenchyme of mouse limb buds. Although most mesenchymal cells respond rapidly to SHH signalling, the transcriptional upregulation of specific SHH target signals in the mesenchyme occurs with differential temporal kinetics and in a spatially restricted fashion. In particular, the expression of the BMP antagonist Grem1 is always upregulated in mesenchymal cells located distal to the SHH source and acts upstream of FGF signalling by the apical ectodermal ridge. GREM1/FGF-mediated feedback signalling is, in turn, required to propagate SHH and establish the presumptive digit expression domains of the Notch ligand jagged 1 (Jag1) and 5'Hoxd genes in the distal limb bud mesenchyme. Their establishment is significantly delayed in Grem1-deficient limb buds and cannot be rescued by specific restoration of SHH signalling in mutant limb buds. This shows that GREM1/FGF feedback signalling is required for regulation of the temporal kinetics of the mesenchymal response to SHH signalling. Finally, inhibition of SHH signal transduction at distinct time points reveals the differential temporal dependence of Grem1, Jag1 and 5'Hoxd gene expression on SHH signalling. In particular, the expression of Hoxd13 depends on SHH signal transduction significantly longer than does Hoxd11 expression, revealing that the reverse co-linear establishment, but not maintenance of their presumptive digit expression domains, depends on SHH signalling.

Hyped‐up for Hox in Hyderabad

The EMBO Workshop on Upstream and Downstream of Hox Genes took place in Hyderabad, India, between 14 and 17 December 2005, and was organized by R. Mishra and F. Karch. ![][1] For many years, EMBO workshops dealing with new insights into the regulation and function of Hox genes have been held in Switzerland. However, last year, the EMBO World Programme, together with the Centre for Cellular and Molecular Biology (CCMB), held a workshop entitled Upstream and Downstream of Hox Genes in the CCMB's hometown of Hyderabad, India. Imagine beautiful gardens full of butterflies and palm trees, a delightful guest house neighbouring a state‐of‐the‐art research centre, all in a peaceful oasis surrounded by a pulsating and crowded Indian city. The question at the beginning of the meeting was whether the talks and discussions would match the stunning environment; it was answered rapidly and affirmatively. Hox genes have had an illustrious and curious history. Perhaps one of the most famous images in biology from the past century is the four‐winged mutant of the fruit fly Drosophila melanogaster caused by mutations in the Hox gene Ultrabithorax ( Ubx ). This fascinating mutant fly, discovered and characterized by Edward B. Lewis, has motivated many investigators to join the field to understand how these genes are responsible for such complex changes in morphology. Another highlight of Hox history was Matthew Scott's, William McGinnis's and Walter Gehring's discovery of the homeodomain—a 60‐amino‐acid DNA‐binding motif now known to be a key domain in hundreds of genes throughout the animal, fungal and plant kingdoms. Although a lot has been learned through experiments stemming from these important discoveries, there are still many open questions. Among the questions being debated in the field and at the meeting are: How is the regulation of the Hox genes initiated and maintained? How do Hox proteins … [1]: /embed/graphic-1.gif

Group 13 HOX proteins interact with the MH2 domain of R-Smads and modulate Smad transcriptional activation functions independent of HOX DNA-binding capability

Interactions with co-factors provide a means by which HOX proteins exert specificity. To identify candidate protein interactors of HOXA13, we created and screened an E11.5–E12.5, distal limb bud yeast two-hybrid prey library. Among the interactors, we isolated the BMP-signaling effector Smad5, which interacted with the paralogous HOXD13 but not with HOXA11 or HOXA9, revealing unique interaction capabilities of the AbdB-like HOX proteins. Using deletion mutants, we determined that the MH2 domain of Smad5 is necessary for HOXA13 interaction. This is the first report demonstrating an interaction between HOX proteins and the MH2 domain of Smad proteins. HOXA13 and HOXD13 also bind to other BMP and TGF-β/Activin-regulated Smad proteins including Smad1 and Smad2, but not Smad4. Furthermore, HOXD13 could be co-immunoprecipitated with Smad1 from cells. Expression of HOXA13, HOXD13 or a HOXD13 homeodomain mutant (HOXD13IQN>AAA) antagonized TGF-β-stimulated transcriptional activation of the pAdtrack-3TP-Lux reporter vector in Mv1Lu cells as well as the Smad3/Smad4-activated pTRS6-E1b promoter in Hep3B cells. Finally, using mammalian one-hybrid assay, we show that transcriptional activation by a GAL4/Smad3-C-terminus fusion protein is specifically inhibited by HOXA13. Our results identify a new co-factor for HOX group 13 proteins and suggest that HOX proteins may modulate Smad-mediated transcriptional activity through protein–protein interactions without the requirement for HOX monomeric DNA-binding capability.

Expression ofHoxa‐11andHoxa‐13in the pectoral fin of a basal ray‐finned fish,Polyodon spathula: implications for the origin of tetrapod limbs

Summary Paleontological and anatomical evidence suggests that the autopodium (hand or foot) is a novel feature that distinguishes limbs from fins, while the upper and lower limb (stylopod and zeugopod) are homologous to parts of the sarcopterygian paired fins. In tetrapod limb development Hoxa-11 plays a key role in differentiating the lower limb and Hoxa-13 plays a key role in differentiating the autopodium. It is thus important to determine the ancestral functions of these genes in order to understand the developmental genetic changes that led to the origin of the tetrapod autopodium. In particular it is important to understand which features of gene expression are derived in tetrapods and which are ancestral in bony fishes. To address these questions we cloned and sequenced the Hoxa-11 and Hoxa-13 genes from the North American paddlefish, Polyodon spathula, a basal ray-finned fish that has a pectoral fin morphology resembling that of primitive bony fishes ancestral to the tetrapod lineage. Sequence analysis of these genes shows that they are not orthologous to the duplicated zebrafish and fugu genes. This implies that the paddlefish has not duplicated its HoxA cluster, unlike zebrafish and fugu. The expression of Hoxa-11 and Hoxa-13 in the pectoral fins shows two main phases: an early phase in which Hoxa-11 is expressed proximally and Hoxa-13 is expressed distally, and a later phase in which Hoxa-11 and Hoxa-13 broadly overlap in the distal mesenchyme of the fin bud but are absent in the proximal fin bud. Hence the distal polarity of Hoxa-13 expression seen in tetrapods is likely to be an ancestral feature of paired appendage development. The main difference in HoxA gene expression between fin and limb development is that in tetrapods (with the exception of newts) Hoxa-11 expression is suppressed by Hoxa-13 in the distal limb bud mesenchyme. There is, however, a short period of limb bud development where Hoxa-11 and Hoxa-13 overlap similarly to the late expression seen in zebrafish and paddlefish. We conclude that the early expression pattern in tetrapods is similar to that seen in late fin development and that the local exclusion by Hoxa-13 of Hoxa-11 from the distal limb bud is a derived feature of limb developmental regulation.

Conserved expression domains for genes upstream and within the HoxA and HoxD clusters suggests a long‐range enhancer existed before cluster duplication

The posterior HoxA and HoxD genes are essential in appendicular development. Studies have demonstrated that a "distal limb enhancer," remotely located upstream of the HoxD complex, is required to drive embryonic autopod expression of the posterior Hox genes as well as the two additional non-Hox genes in the region: Evx2 and Lnp. Our work demonstrates a similar mode of regulation for Hoxa13 and four upstream genes: Evx1, Hibadh, Tax1bp, and Jaz1. These genes all show embryonic (E11.5-E13.5) distal limb and genital bud expression, suggesting the existence of a nearby enhancer influencing the expression of a domain of genes. Comparative sequence analysis between homologous human and mouse genomic sequence upstream of Hoxa13 revealed a remote 2.25-kb conserved noncoding sequence (mmA13CNS) within the fourth intron of the Hibadh gene. mmA13CNS shares a common 131-bp core identity within a conserved noncoding sequence upstream of Hoxd13, which is located within the previously identified distal limb enhancer critical region. To test the function of this conserved sequence, we created mmA13CNS-Hsp86-lacZ transgenic mice. mmA13CNS directed a wide range of tissue expression, including the central nervous system, developing olfactory tissue, limb, and genital bud. Limb and genital bud expression directed by mmA13CNS is not identical to the patterns exhibited by Hoxa13/Evx1/Hibadh/Tax1bp1/Jaz1, suggesting that mmA13CNS is not sufficient to fully recapitulate their expression in those tissues. The Evx1- and Evx2-like central nervous system expression observed in these mice suggests that the long-range regulatory element(s) for the Hox cluster existed before the cluster duplication.

Wnt Versus Wnt

In the canonical Wnt signaling pathway, Wnts cause stabilization of β-catenin and consequent changes in gene expression. But not all Wnts appear to signal in this way. Developmental actions of Wnt-5, for example, appear not to be mediated by β-catenin, and now two papers show that Wnt-5 actually antagonizes canonical Wnt signaling. Westfall et al. explored developmental roles of WNt-5 in zebrafish. The dorsalization defects they observed when expression of maternal and zygotic Wnt-5 was removed were similar to effects of activation of canonical Wnt-β-catenin signaling and were associated with accumulation of β-catenin. Loss of Wnt-5 caused decreased Ca 2+ release, and overexpression of Ca 2+ -calmodulin-dependent protein kinase II partially rescued the effects of Wnt-5 mutation. Thus, the authors conclude that Wnt-5 actually antagonizes the effects of canonical Wnts, possibly by a Ca 2+ -mediated signal. Topol et al. offer an alternative mechanism. In cultured mammalian 293 cells and in a human colon cancer cell line, ectopic expression of Wnt-5a antagonized canonical Wnt signaling and caused degradation of β-catenin. Their analysis, however, indicated that Wnt-5's effects were largely independent of Ca 2+ -mediated signals. Rather, they propose that Wnt-5a may increase expression of Siah2, a component of the proteasome, which mediates degradation of β-catenin. In 293 cells, dominant-negative Siah2 reduced the effects of Wnt-5a on β-catenin signaling, and ectopic expression of Wnt-5a increased expression of Siah2. In Wnt-5a knockout mice, β-catenin also accumulated in the distal limb bud during development. Thus, in limb buds lacking Wnt-5a, unchecked canonical Wnt signaling may cause developmental abnormalities. Consistent with this idea, chondorgenesis is inhibited in Wnt5a –/– limbs, but was partially rescued by grafted chick embryonic fibroblast cells engineered to express a secreted antagonist of Wnt signaling. Both groups agree that proper development appears to require a balance of opposing Wnt signals and that disruption of that balance could contribute to abnormal canonical Wnt signaling, which is implicated in formation of some human cancers. T. A. Westfall, R. Brimeyer, J. Twedt, J. Gladon, A. Olberding, M. Furutani-Seiki, D. C. Slusarski, Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator of Wnt/β-catenin activity. J. Cell Biol. 162 , 889-898 (2003). [Abstract] [Full Text] L. Topol, X. Jiang, H. Choi, L. Garrett-Beal, P. J. Carolan, Y. Yang, Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent β-catenin degradation. J. Cell Biol. 162 , 899-908 (2003). [Abstract] [Full Text]

When Wnts antagonize Wnts.

Secreted Wnt ligands appear to activate a variety of signaling pathways. Two papers in this issue now present genetic evidence that “noncanonical” Wnt signaling inhibits the “canonical” Wnt/β-catenin pathway. Westfall et al. (2003a) show that zebrafish embryos lacking maternal Wnt-5 function are dorsalized due to ectopic activation of β-catenin, whereas Topol et al. (2003) report that chondrogenesis in the distal mouse limb bud depends on inhibition of Wnt/β-catenin signaling by a paralogue of Wnt-5. These studies present the first genetic confirmation of the previous hypothesis that vertebrate Wnt signaling pathways can act in an antagonistic manner.

Nonmuscle myosin promotes cytoplasmic localization of PBX.

In the absence of MEIS family proteins, two mechanisms are known to restrict the PBX family of homeodomain (HD) transcription factors to the cytoplasm. First, PBX is actively exported from the nucleus via a CRM1-dependent pathway. Second, nuclear localization signals (NLSs) within the PBX HD are masked by intramolecular contacts. In a screen to identify additional proteins directing PBX subcellular localization, we identified a fragment of murine nonmuscle myosin II heavy chain B (NMHCB). The interaction of NMHCB with PBX was verified by coimmunoprecipitation, and immunofluorescence staining revealed colocalization of NMHCB with cytoplasmic PBX in the mouse embryo distal limb bud. The interaction domain in PBX mapped to a conserved PBC-B region harboring a potential coiled-coil structure. In support of the cytoplasmic retention function, the NMHCB fragment competes with MEIS1A to redirect PBX, and the fly PBX homologue EXD, to the cytoplasm of mammalian and insect cells. Interestingly, MEIS1A also localizes to the cytoplasm in the presence of the NMHCB fragment. These activities are largely independent of nuclear export. We show further that the subcellular localization of EXD is deregulated in Drosophila zipper mutants that are depleted of nonmuscle myosin heavy chain. This study reveals a novel and evolutionarily conserved mechanism controlling the subcellular distribution of PBX and EXD proteins.

Mouse Twist is required for fibroblast growth factor-mediated epithelial–mesenchymal signalling and cell survival during limb morphogenesis

Mouse Twist is essential for cranial neural tube, limb and somite development. [Genes Dev. 9 (1995) 686]. To identify the molecular defects disrupting limb morphogenesis, we have analysed expression of mesenchymal transcription factors involved in patterning and the cell–cell signalling cascades controlling limb bud development. These studies establish that Twist is essential for maintenance and progression of limb bud morphogenesis. In particular, the SHH/FGF signalling feedback loop operating between the polarizing region and the apical ectodermal ridge (AER) is disrupted. These defects in epithelial–mesenchymal signalling are most likely a direct consequence of disrupted fibroblast growth factor (FGF) signalling in Twist-deficient limb buds. In early limb buds, down-regulation of Fgf receptor 1 and Fgf10 expression in the mesenchyme occurs concurrent with loss of Fgf4 and Fgf8 expression in the AER. Finally, Twist function, most likely by regulating FGF signalling, is required for cell survival as apoptotic cells are detected in posterior and distal limb bud mesenchyme.

Retinoic acid specifically downregulates Fgf4 and inhibits posterior cell proliferation in the developing mouse autopod.

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.

Evidence that preaxial polydactyly in the Doublefoot mutant is due to ectopic Indian Hedgehog signaling.

Patterning of the vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) located at the posterior limb margin. One of the vertebrate Hh family members, Shh, has been shown to be able to mediate the function of the ZPA. Several naturally occurring mouse mutations with the phenotype of preaxial polydactyly exhibit ectopic Shh expression at the anterior limb margin. In this study, we report the molecular characterization of a spontaneous mouse mutation, Doublefoot (Dbf). Dbf is a dominant mutation which maps to chromosome 1. Heterozygous and homozygous embryos display a severe polydactyly with 6 to 8 digits on each limb. We show here that Shh is expressed normally in Dbf mutants. In contrast, a second Hh family member, Indian hedgehog (Ihh) which maps close to Dbf, is ectopically expressed in the distal limb bud. Ectopic Ihh expression in the distal and anterior limb bud results in the ectopic activation of several genes associated with anterior-posterior and proximal-distal patterning (Fgf4, Hoxd13, Bmp2). In addition, specific components in the Hedgehog pathway are either ectopically activated (Ptc, Ptc-2, Gli1) or repressed (Gli2). We propose that misexpression of Ihh, and not a novel Smoothened ligand as recently suggested (Hayes et al., 1998), is responsible for the Dbf phenotype. We consider that Ihh has a similar activity to Shh when expressed in the early Shh-responsive limb bud. To determine whether Dbf maps to the Ihh locus, which is also on chromosome 1, we performed an interspecific backcross. These results demonstrate that Dbf and Ihh are genetically separated by approximately 1.3 centimorgans, suggesting that Dbf mutation may cause an exceptionally long-range disruption of Ihh regulation. Although this leads to ectopic activation of Ihh, normal expression of Ihh in the cartilaginous elements is retained.

Differential Expression of the Two Closely Related LIM-Class Homeobox GenesLH-2AandLH-2Bduring Limb Development

We have isolated the chicken homeobox genesLH-2AandLH-2Bencoding two related LIM domain-containing homeodomain proteins and examined the expression pattern during chick limb development.LH-2Ais most closely related to human and ratLH-2,whileLH-2Bis less conserved. Although bothLH-2AandLH-2Bare expressed in the limb mesenchyme throughout stage 16 to stage 32,LH-2Atranscripts are detectable in the distal limb bud andLH-2Btranscripts are detectable in the anterior limb bud. Signals from the apical ectodermal ridge positively regulateLH-2Aexpression, since removal of the apical ectoderm resulted in the rapid reduction ofLH-2Aexpression in the distal limb mesenchyme. Ectopic expression of thesonic hedgehoggene in the anterior margin of the limb bud resulted in the rapid reduction ofLH-2Bexpression accompanying respecification of the positional value to the posterior phenotype. These results suggest thatLH-2AandLH-2Bplay important roles in the determination and specification of the proximal-distal and anterior-posterior positional values, respectively.

The Apert syndrome hand: pathologic anatomy and clinical manifestations.

The Apert syndrome hand demonstrates many typical clinical features including syndactyly, symbrachyphalangism, and growth disturbances. This is due to the grossly abnormal anatomy of both the skeletal and soft-tissue structures associated with a progressive disease process. This paper presents a clinical, radiologic, and histologic analysis of the Apert syndrome hand anatomy and correlates it with the clinical manifestations. It also links hand and craniofacial dysplasia to other regions of the skeleton as well as the overall disease process. From our analysis, we conclude that there is a genetic anomaly causing variable and uncoordinated differentiation of the mesenchyme at the time of embryologic separation into its various skeletal components, particularly in the distal limb bud and craniofacial skeleton. This disease process continues postnatally in endochondral bone growth center malformation and malfunction as well as ectopic cartilage ossification in soft tissues. We discuss the role of abnormal musculotendinous anatomy and altered biomechanical forces in relation to these processes.

Insulin and Insulin-like Growth Factor I Prevent the Mitogenic Response of Chick Limb Bud Mesoderm Cells to Platelet-Derived Growth Factor-AA

Platelet-derived growth factor AA (PDGF-AA) increases DNA synthesis by chicken limb bud mesoderm in culture. Preincubation of either mesoderm from whole limb buds (stage 24-25) or mesoderm from the distal tip of the bud (stage 25) in insulin or insulin-like growth factor type I (IGF-I) resulted in increased DNA synthesis compared to control levels in two different types of medium. However, no further increase in DNA synthesis was then induced by PDGF-AA. This was found even when the level of DNA synthesis in IGF-I or insulin-containing medium was below that seen in cells treated only with PDGF. The response to PDGF was not dependent on the presence of serum in the medium. Insulin and IGF-I inhibited expression of the PDGF α receptor in mesoderm from whole limb buds. However, this was detected at times after the increase in DNA synthesis in response to PDGF was normally seen. IGF-I did not inhibit expression of the PDGF α receptor in mesoderm from distal limb bud tips. Thus, IGF-I and insulin modify the response of limb bud mesoderm to PDGF. The effects of IGF-I and insulin on receptor expression may be dependent on the cell populations present but do not appear to account for modulation of the PDGF-induced mitogenic response.