A gradient of homeodomain protein in developing forelimbs of Xenopus and mouse embryos

The expression of the homeodomain protein XIHbox 1 in developing Xenopus limbs was analyzed using specific antibodies. In the forelimb bud mesoderm XIHbox 1 shows a clear antero-posterior gradient that is strongest in the anterior and proximal region of the forelimb. Hindlimb bud mesoderm is devoid of XIHbox 1, indicating an early molecular difference between arm and leg. The innermost ectodermal cell layer is positive throughout the forelimb and hindlimb bud ectoderm, but no other areas of the skin. Similar results are obtained in developing mouse limbs, suggesting that XIHbox 1 participates in forelimb development in a variety of tetrapods. In early tadpoles analyzed at stages preceding limb bud formation, the lateral plate mesoderm is positive in the region corresponding to the earliest "field" of forelimb information, but not in the hindlimb field. These results suggest a molecular link between morphogenetic fields, gradients, and homeobox genes in vertebrate development.     

The dissociation of the Fgf-feedback loop controls the limbless state of the neck

In tetrapods, limbs develop at two specific positions along the anteroposterior axis of the embryo, whereas other regions of the embryo, most prominently the neck and the flank, are limbless. However, the flank can generate an ectopic limb when the Fgf-feedback loop crucial for the initiation of limb budding is activated. Thus, despite its limblessness, the flank is a limb-competent area. Using the chick embryo as model, we investigated whether the neck, as the flank, has the competence to form a limb, and what mechanism may regulate its limblessness. We show that forelimb lateral mesoderm plus ectoderm grafted into the neck can continue limb development, suggesting that the neck does not actively inhibit this process. However, neck tissues themselves do not support or take part in limb formation. Hence, the neck is limb-incompetent. This is due to the dismantling of Fgf signalling at distinct points of the MAPK signalling cascade in the neck lateral mesoderm and ectoderm.     

The nuclear orphan receptor COUP-TFII is required for limb and skeletal muscle development

The nuclear orphan receptor COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development, suggesting that COUP-TFII is involved in multiple aspects of embryogenesis. Because of the early embryonic lethality of COUP-TFII knockout mice, the role of COUP-TFII during limb development has not been determined. COUP-TFII is expressed in lateral plate mesoderm of the early embryo prior to limb bud formation. In addition, COUP-TFII is also expressed in the somites and skeletal muscle precursors of the limbs. Therefore, in order to study the potential role of COUP-TFII in limb and skeletal muscle development, we bypassed the early embryonic lethality of the COUP-TFII mutant by using two methods. First, embryonic chimera analysis has revealed an obligatory role for COUP-TFII in limb bud outgrowth since mutant cells are unable to contribute to the distally growing limb mesenchyme. Second, we used a conditional-knockout approach to ablate COUP-TFII specifically in the limbs. Loss of COUP-TFII in the limbs leads to hypoplastic skeletal muscle development, as well as shorter limbs. Taken together, our results demonstrate that COUP-TFII plays an early role in limb bud outgrowth but not limb bud initiation. Also, COUP-TFII is required for appropriate development of the skeletal musculature of developing limbs.     

Expression of lbx1 involved in the hypaxial musculature formation of the mouse embryo

Somites are the source of hypaxial musculature including skeletal muscles of the limb, tongue, and trunk. To get insight into the function of mouse Lbx1 homeobox gene in early somitic mesoderm differentiation, in situ hybridization analyses were performed. At the 4-6 somite stage (8 dpc), Lbx1 was first expressed in the lateral portion of the epithelial somite and dermomyotomal epithelium. This was in contrast to the expression of myf-5 in the medial region of the somite. The lateral expression of Lbx1 in somitic mesoderm then occurred regionally along the anterior-posterior body axis. Later, at 10 dpc (stage 1 of limb bud development), Lbx1-positive migrating cells originated in the lateral dermomyotomal lips at occipital, forelimb, and hindlimb levels. They also expressed Pax-3 and c-met, known as markers of the migrating limb muscle precursor cells. In stage 4 hindlimb bud (11.5 dpc), the dorsal and ventral muscle precursor populations expressed Lbx1. In stage 8 forelimb buds (12.5 dpc), Lbx1 expression was reduced in the proximal muscle masses, where the high expression of myogenin accompanying muscle differentiation was detected. These results suggest that mouse Lbx1 might be involved in the commitment or determination of a muscle cell subpopulation during hypaxial musculature development. J. Exp. Zool. 286:270-279, 2000.     

Disrupting the establishment of polarizing activity by teratogen exposure.

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.     

Tbx5 and Tbx4 are not sufficient to determine limb-specific morphologies but have common roles in initiating limb outgrowth

Morphological differences between forelimbs and hindlimbs are thought to be regulated by Tbx5 expressed in the forelimb and Tbx4 and Pitx1 expressed in the hindlimb. Gene deletion and misexpression experiments have suggested that these factors have two distinct functions during limb development: the initiation and/or maintenance of limb outgrowth and the specification of limb-specific morphologies. Using genetic methods in the mouse, we have investigated the roles of Tbx5, Tbx4, and Pitx1 in both processes. Our results support a role for Tbx5 and Tbx4, but not for Pitx1, in initiation of limb outgrowth. In contrast to conclusions from gene misexpression experiments in the chick, our results demonstrate that Tbx5 and Tbx4 do not determine limb-specific morphologies. However, our results support a role for Pitx1 in the specification of hindlimb-specific morphology. We propose a model in which positional codes, such as Pitx1 and Hox genes in the lateral plate mesoderm, dictate limb-specific morphologies.     

Retinoic acid synthesis controlled by Raldh2 is required early for limb bud initiation and then later as a proximodistal signal during apical ectodermal ridge formation

We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2(-/-) embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2(-/-) forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.     

WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo

A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Here, we show that three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm and the lateral plate mesoderm in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, we also show that the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo.     

A comparative analysis of Meox1 and Meox2 in the developing somites and limbs of the chick embryo

We have examined the expression pattern of the avian Meox1 homeobox gene during early development and up to late limb bud stages. Its expression pattern indicates that it is involved in somite specification and differentiation. The domains of expression are similar but different to those of Meox2. Meox1 is expressed from stage 6 in the pre-somitic mesoderm and as development proceeds, in the tail bud, the dermomyotome of the rostral somites and in the dermomyotome and sclerotome of the caudal somites, the lateral rectus muscle, truncus arteriosus of the heart and the limb buds. Unlike Meox1, Meox2 is not expressed in the pre-somitic mesoderm, but is expressed first in somites formed from stage 11 onwards. In the developing limb, both genes are expressed in the dorsal and ventral limb mesoderm in adjacent domains with a small region of overlap. In the limb bud, Meox1 is co-expressed with Meox2 but neither Meox gene is co-expressed with MyoD. These expression patterns suggest that these two genes have overlapping and distinct functions in development.     

Mesodermal and neuronal retinoids regulate the induction and maintenance of limb innervating spinal motor neurons

During embryonic development, the generation, diversification and maintenance of spinal motor neurons depend upon extrinsic signals that are tightly regulated. Retinoic acid (RA) is necessary for specifying the fates of forelimb-innervating motor neurons of the Lateral Motor Column (LMC), and the specification of LMC neurons into medial and lateral subtypes. Previous studies implicate motor neurons as the relevant source of RA for specifying lateral LMC fates at forelimb levels. However, at the time of LMC diversification, a significant amount of retinoids in the spinal cord originates from the adjacent paraxial mesoderm. Here we employ mouse genetics to show that RA derived from the paraxial mesoderm is required for lateral LMC induction at forelimb and hindlimb levels, demonstrating that mesodermally synthesized RA functions as a second source of signals to specify lateral LMC identity. Furthermore, reduced RA levels in postmitotic motor neurons result in a decrease of medial and lateral LMC neurons, and abnormal axonal projections in the limb; invoking additional roles for neuronally synthesized RA in motor neuron maintenance and survival. These findings suggest that during embryogenesis, mesodermal and neuronal retinoids act coordinately to establish and maintain appropriate cohorts of spinal motor neurons that innervate target muscles in the limb.     

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.     

Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression

Vertebrate limb development occurs along three cardinal axes-proximodistal, anteroposterior and dorsoventral-that are established via the organization of signaling centers, such as the zone of polarizing activity (ZPA). Distal limb development, in turn, requires a molecular feedback loop between the ZPA expression of sonic hedgehog (Shh) and the apical ectodermal ridge. The TALE homeoprotein Pbx1 has been shown to be essential for proximal limb development. In this study, we first uncover that Pbx1 and Pbx2 are co-expressed in the lateral plate and early limb field mesoderm. Later, Pbx2 is expressed throughout the limb, unlike Pbx1, which is expressed only in the proximal bud. By exploiting a Pbx1/Pbx2 loss-of-function mouse model, we demonstrate that, despite the lack of limb abnormalities in Pbx2-deficient (Pbx2(-/-)) embryos, compound Pbx1(-/-); Pbx2(+/-) mutants, in addition to their exacerbated proximal limb defects, exhibit novel and severe distal abnormalities. Additionally, we reveal that Pbx1(-/-); Pbx2(-/-) embryos lack limbs altogether. Furthermore, we establish that, unlike in flies, where the leg develops independently of Hox and where the Pbx ortholog Exd is required for specification of proximal (but not distal) limbs, in vertebrates, distal limb patterning is Pbx1/Pbx2 dependent. Indeed, we demonstrate that Pbx genetic requirement is mediated, at least in part, through their hierarchical control of Hox spatial distribution and Shh expression. Overall, we establish that, by controlling the spatial expression of Hox genes in the posterior limb and regulating ZPA function, Pbx1/Pbx2 exert a primary hierarchical function on Hox genes, rather than behaving merely as Hox ancillary factors.     

Differential Distribution of Retinoic Acid Synthesis in the Chicken Embryo as Determined by Immunolocalization of the Retinoic Acid Synthetic Enzyme, RALDH-2

Retinaldehyde dehydrogenase type 2 (RALDH-2) is a major retinoic acid generating enzyme in the early embryo. Here we report the immunolocalization of this enzyme (RALDH-2-IR) in stage 6-29 chicken embryos; we also show that tissues that exhibit strong RALDH-2-IR in the embryo contain RALDH-2 and synthesize retinoic acid. RALDH-2-IR indicates dynamic and discrete patterns of retinoic acid synthesis in the embryo, particularly within the somitic mesoderm, lateral mesoderm, kidney, heart, and spinal motor neurons. Prior to somitogenesis, RALDH-2-IR is present in the paraxial mesoderm with a rostral boundary at the level of the presumptive first somite; as the somites form, they exhibit strong RALDH-2-IR. Cervical presomitic mesoderm exhibits RALDH-2-IR but thoracic presomitic mesoderm does not. Neural crest cells do not express detectable levels of RALDH-2, but migrating crest cells are associated with RALDH-2 expressing mesoderm. The developing limb mesoderm expresses little RALDH-2-IR; however, RALDH-2-IR is strongly expressed in tissues adjacent to the limb. The most lateral, earliest-projecting motor neurons at all levels of the spinal cord exhibit RALDH-2-IR. Subsequently, many additional motor neurons in the brachial and lumbar cord regions express RALDH-2-IR. Motor neuronal expression of RALDH-2-IR is present in the growing axons as they extend to the periphery, indicating a potential role of retinoic acid in nerve influences on peripheral differentiation. With the exception of a transient expression in the facial/vestibulocochlear nucleus, cranial motor neurons do not express detectable levels of RALDH-2-IR.