Level-specific role of paraxial mesoderm in regulation of Tbx5/Tbx4 expression and limb initiation

Tetrapod limbs, forelimbs and hindlimbs, emerge as limb buds during development from appropriate positions along the rostro-caudal axis of the main body. In this study, tissue interactions by which rostro-caudal level-specific limb initiation is established were analyzed. The limb bud originates from the lateral plate located laterally to the paraxial mesoderm, and we obtained evidence that level-specific tissue interactions between the paraxial mesoderm and the lateral plate mesoderm are important for the determination of the limb-type-specific gene expression and limb outgrowth. When the wing-level paraxial mesoderm was transplanted into the presumptive leg region, the wing-level paraxial mesoderm upregulated the expression of Tbx5, a wing marker gene, and down regulated the expression of Tbx4 and Pitx1, leg marker genes, in the leg-level lateral plate. The wing-level paraxial mesoderm relocated into the leg level also inhibited outgrowth of the hindlimb bud and down regulated Fgf10 and Fgf8 expression, demonstrating that the wing-level paraxial mesoderm cannot substitute for the function of the leg-level paraxial mesoderm in initiation and outgrowth of the hindlimb. The paraxial mesoderm taken from the neck- and flank-level regions also had effects on Tbx5/Tbx4 expression with different efficiencies. These findings suggest that the paraxial mesoderm has level-specific abilities along the rostro-caudal axis in the limb-type-specific mechanism for limb initiation.     

Tbx4 is not required for hindlimb identity or post-bud hindlimb outgrowth

Tbx4 is a crucial gene in the initiation of hindlimb development and has been reported as a determinant of hindlimb identity and a presumptive direct regulator of Fgf10 in the limb. Using a conditional allele of Tbx4, we have ablated Tbx4 function before and after limb initiation. Ablation of Tbx4 before expression in the hindlimb field confirms its requirement for limb bud outgrowth. However, ablation of Tbx4 shortly after onset of expression in the hindlimb field, during limb bud formation, alters neither limb outgrowth nor expression of Fgf10. Instead, post-limb-initiation loss of Tbx4 results in reduction of limb core tissue and hypoplasia of proximal skeletal elements. Loss of Tbx4 during later limb outgrowth produces no limb defects, revealing a brief developmental requirement for Tbx4 function. Despite evidence from ectopic expression studies, our work establishes that loss of Tbx4 has no effect on hindlimb identity as assessed by morphology or molecular markers.     

Medaka unextended-fin mutants suggest a role for Hoxb8a in cell migration and osteoblast differentiation during appendage formation

Hoxb8 has been suggestively implicated in the formation of the zone of polarizing activity (ZPA) in the limb bud. However, as hoxb8-/- mice did not show any defects in their limb development, the role of Hoxb8 during limb development has not been fully elucidated. Here, we report the identification of the medaka hoxb8a mutant, unextended-fin (ufi), in which all the fin tissues were malformed. Since the abnormal phenotype was observed in the caudal fin, the ufi phenotype suggests that the medaka Hoxb8a has a fundamental role in the formation of appendages protruding from the trunk. Our analyses revealed that the expression of wnt5a, a regulator of cell migration that signals through the non-canonical Wnt/Ca2+ pathway, was down-regulated in the ufi fin-folds. In fact, we found that the proximal-distal cell migration was impaired in ufi mutants and that the defect could be reversed by the injection of a Wnt5a protein. Moreover, we show herein that the numbers of proliferating cells and osteoblastic cells were increased in the ufi mutants. According to these results, we propose that the medaka Hoxb8a protein functions in the outgrowth of appendages through the regulation of cell migration and osteoblast differentiation.     

Fibin, a novel secreted lateral plate mesoderm signal, is essential for pectoral fin bud initiation in zebrafish

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.     

The microRNA-processing enzyme Dicer is dispensable for somite segmentation but essential for limb bud positioning

Dicer is an enzyme that processes microRNAs (miRNAs) to their mature forms. As miRNAs were first discovered for their role in the control of developmental timing, we investigated their potential requirement in mouse somitogenesis, an event with precise temporal periodicity. To address the collective role of miRNAs in mesoderm development including somite formation, we used T (Brachyury)-Cre mouse line to inactivate Dicer in most cells of the mesoderm lineage. This Dicer mutant exhibits a reduced anterior–posterior axis. Somite number remains normal in mutant embryos up until the death of the embryos more than two days after Dicer inactivation. Consistent with this, the molecular machineries required for establishing segmentation, including clock and wave front, are not perturbed. However, somite size is reduced and later-formed somites are caudalized, coincident with increased cell death. Outside of the paraxial mesoderm and prior to apparent reduction of the axis in the mutant, the position of the hindlimb bud, a lateral plate mesoderm-derived structure, is posteriorly shifted and the timing of hindlimb bud initiation is delayed accordingly. We observed changes in the expression of genes critical for limb positioning, which include a shifted and delayed downregulation of Hand2 and Tbx3, and shifted and delayed upregulation of Gli3 in the prospective limb bud field. The 3′ UTRs of both Hand2 and Tbx3 harbor target sites for a seed sequence-sharing family of miRNAs mir-25/32/92/363/367. As an example of the family we show that mir-363, a miRNA with elevated expression in the prospective limb bud field, is capable of inhibiting Hand2/Tbx3 expression in vitro in a binding site-dependent manner. Together, our findings provide the first demonstration that in mouse embryonic mesoderm, while Dicer is dispensable for somite segmentation, it is essential for proper limb bud positioning.     

Tbx18 and boundary formation in chick somite and wing development

The chicken Tbx gene, Tbx18, is expressed in lateral plate mesoderm, limb, and developing somites. Here we show that Tbx18 is expressed transiently in axial mesenchyme during somite segmentation. We present evidence from overexpression and transplantation experiments that Tbx18 controls fissure formation in the late stages of somite maturation. In presumptive wing lateral plate mesoderm, ectopic Tbx18 expression leads to anterior extension of the wing bud. These results suggest that Tbx18 is involved in producing mesodermal boundaries, generating in paraxial mesoderm morphological boundaries between somites and in lateral plate mesoderm a wing- or non-wing-forming boundary.     

Tbx2 Terminates Shh/Fgf Signaling in the Developing Mouse Limb Bud by Direct Repression of Gremlin1

Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.     

Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.     

Notch1 and 2 cooperate in limb ectoderm to receive an early Jagged2 signal regulating interdigital apoptosis

Spontaneous and engineered mutations in the Notch ligand Jagged2 produced the Syndactylism phenotype (Jiang, R.L., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G., Gridley, T., 1998. Defects in limb, craniofacial, and thymic Development in Jagged2 mutant mice. Genes Dev. 12, 1046-1057; Sidow, A., Bulotsky, M.S., Kerrebrock, A.W., Bronson, R.T., Daly, M.J., Reeve, M.P., Hawkins, T.L., Birren, B.W., Jaenisch, R., Lander, E.S., 1997. Serrate2 is disrupted in the mouse limb-development mutant syndactylism. Nature 389, 722-725). Given that additional ligands may be expressed in the developing limb bud, it was possible that loss of Jagged2 disabled only part of Notch function in the limb. In addition, it is not clear from the expression pattern of Jagged2 in the apical ectodermal ridge (AER) whether the ectodermal or mesenchymal compartment of the limb bud receives the Jagged2 signal. To elucidate the requirement for the Notch pathway in limb development, we have analyzed single and compound Notch receptor mutants as well as gamma-secretase-deficient limbs. Floxed alleles were removed either from the developing limb bud ectoderm (using Msx2-Cre) or from the mesenchyme (using Prx1-Cre). Our results confirm that Jagged2 loss describes the contribution of the entire Notch pathway to the mouse limb development and revealed that both Notch1 and 2 are required in the ectoderm to receive the Jagged2 signal. Interestingly, our allelic series allowed us to determine that Notch receives this signal at an early stage in the developmental process and that memory of this event is retained by the mesenchyme, where Notch signaling appears to be dispensable. Thus, Notch signaling plays a non-autonomous role in digit septation.     

The clonal composition of biramous and uniramous arthropod limbs

We present the first comparative cell lineage analysis of uniramous and biramous limbs of an arthropod, the crustacean Orchestia cavimana. Via single cell labelling of the cells that are involved in limb development, we are able to present the first complete clonal composition of an arthropod limb. We show that the two main branches of crustacean limbs, exopod and endopod, are formed by a secondary subdivision of the growth zone of the main limb axis. Additional limb outgrowths such as exites result from the establishment of new axes. In contrast to general belief, uniramous limbs in Orchestia are not formed by the loss of the exopod but by suppression of the split into exopod and endopod. Our results offer a developmental approach to discriminate between the different kinds of branches of arthropod appendages. This leads to the conclusion that a 'true' biramous limb comprising an endopod and an exopod might have occurred much later in euarthropod evolution than has previously been thought, probably either in the lineage of the Mandibulata or that of the Tetraconata.     

Serial homology and the evolution of mammalian limb covariation structure

The tetrapod forelimb and hindlimb are serially homologous structures that share a broad range of developmental pathways responsible for their patterning and outgrowth. Covariation between limbs, which can introduce constraints on the production of variation, is related to the duplication of these developmental factors. Despite this constraint, there is remarkable diversity in limb morphology, with a variety of functional relationships between and within forelimb and hindlimb elements. Here we assess a hierarchical model of limb covariation structure based on shared developmental factors. We also test whether selection for morphologically divergent forelimbs or hindlimbs is associated with reduced covariation between limbs. Our sample includes primates, murines, a carnivoran, and a chiropteran that exhibit varying degrees of forelimb and hindlimb specialization, limb size divergence, and/or phylogenetic relatedness. We analyze the pattern and significance of between-limb morphological covariation with linear distance data collected using standard morphometric techniques and analyzed by matrix correlations, eigenanalysis, and partial correlations. Results support a common limb covariation structure across these taxa and reduced covariation between limbs in nonquadruped species. This result indicates that diversity in limb morphology has evolved without signficant modifications to a common covariation structure but that the higher degree of functional limb divergence in bats and, to some extent, gibbons is associated with weaker integration between limbs. This result supports the hypothesis that limb divergence, particularly selection for increased functional specialization, involves the reduction of developmental factors common to both limbs, thereby reducing covariation.