Interactive Fly, Drosophila

Mouse Engrailed: Function in limb development

Mouse en-1, expressed in embryonic ventral limb ectoderm, is essential for ventral limb patterning. Loss of En-1 results in dorsal transformations of ventral paw structures, and in subtle alterations along the proximal-distal limb axis. EN-1 seems to act in part by repressing dorsal differentiation induced by Wnt-7a, a homolog of Drosophila wingless, and is essential for proper formation of the apical epidermal ridge (Loomis, 1996).

Expression and mutation analyses in mice suggest that the homeobox-containing gene Engrailed is involved in dorsoventral patterning of the limb. En-1 expression is first detected in the flanking ectoderm of the trunk at stage 15; by stage 16, expression extends throughout the length of the ventral body wall. At stage 18, the anterior limit of expression is clearly demarcated at the anterior edge of the wing bud at the level of somite 15. During the initial stages of limb bud outgrowth, En-1 mRNA and protein are uniformly distributed throughout the ventral limb bud ectoderm. Limbs of En-1(-/-) mice display a double dorsal phenotype suggesting that normal expression of En-1 in the ventral ectoderm is required to establish and/or maintain ventral limb characteristics. Loss of En-1 function also results in ventral expansion of the apical ectodermal ridge (AER), suggesting that En-1 is also required for proper formation of the AER. To further investigate the role En plays in dorsoventral patterning and AER formation, the replication competent retroviral vector, RCAS, has been used to mis-express mouse En-1 in the early chick limb bud. Ectopic En-1 expression in dorsal ectoderm is sufficient to repress the endogenous expression of the dorsal ectodermal marker Wnt7a, with a resultant decrease in Lmx1 (See Drosophila Apterous) expression in underlying dorsal mesenchyme. Wnt7a appears to mediate dorsalization of underlying limb mesenchyme through induction of Lmx1, a LIM homeobox gene. The AER is disrupted morphologically and the expression patterns of the AER (ectodermal) signaling molecules Fgf-8 and Fgf-4 are altered. Consistent with recent evidence that there is a reciprocal interaction between signalling molecules in the dorsal ectoderm, AER, and the zone of polarizing activity (ZPA), loss of Wnt7a, Fgf-8 and Fgf-4 expression leads to a decrease in expression of the signalling molecule Shh in the posteriorly positioned ZPA. These results strongly support the idea that in its normal domain of expression, En-1 represses Wnt7a-mediated dorsal differentiation by limiting the expression of Wnt7a to the dorsal ectoderm. These results provide additional evidence that En-1 is involved in AER formation and suggest that En-1 may act to define ventral ectodermal identity (Logan, 1997).

Classical embryological experiments have demonstrated that dorsal-ventral patterning of the vertebrate limb is dependent on ectodermal signals. One such factor is Wnt-7a, a member of the Wnt family of secreted proteins, which is expressed in the dorsal ectoderm. Loss of Wnt-7a results in the appearance of ventral characteristics in the dorsal half of the distal limb. Conversely, En-1, a homeodomain transcription factor, is expressed exclusively in the ventral ectoderm, where it represses Wnt-7a. En-1 mutants have dorsal characteristics in the ventral half of the distal limb. Experiments in the chick suggest that the dorsalizing activity of Wnt-7a in the mesenchyme is mediated through the regulation of the LIM-homeodomain transcription factor Lmx-1. The relationship between Wnt-7a, En-1 and Lmx-1b, a mouse homolog of chick Lmx-1, is examined in the patterning of the mammalian limb. Wnt-7a is required for Lmx-1b expression in distal limb mesenchyme; Lmx-1b activation in the ventral mesenchyme of En-1 mutants requires Wnt-7a. Consistent with Lmx-1b playing a primary role in dorsalization of the limb, a direct correlation is found between regions of the anterior distal limb in which Lmx-lb is misregulated during limb development and the localization of dorsal-ventral patterning defects in Wnt-7a and En-1 mutant adults. Thus, ectopic Wnt-7a expression and Lmx-1b activation underlie the dorsalized En-1 phenotype, although this analysis also reveals a Wnt-7a-independent activity for En-1 in the repression of pigmentation in the ventral epidermis. Ectopic expression of Wnt-7a in the ventral limb ectoderm of En-1 mutants results in the formation of a second, ventral apical ectodermal ridge (AER) at the junction between Wnt-7a-expressing and nonexpressing ectoderm. Unlike the normal AER, ectopic AER formation is dependent upon Wnt-7a activity, indicating that distinct genetic mechanisms may be involved in primary and secondary AER formation (Cygan, 1997).

The apical ectodermal ridge (AER), a rim of thickened ectodermal cells at the interface between the dorsal and ventral domains of the limb bud, is required for limb outgrowth and patterning. The limbs of En1 mutant mice display dorsal-ventral and proximal-distal abnormalities, the latter being reflected in the appearance of a broadened AER and formation of ectopic ventral digits. A detailed genetic analysis of wild-type, En1 and Wnt7a mutant limb buds during AER development has delineated a role for En1 in normal AER formation. These studies support previous suggestions that AER maturation involves the compression of an early broad ventral domain of limb ectoderm into a narrow rim at the tip and show that En1 plays a critical role in the compaction phase. Loss of En1 leads to a delay in the distal shift and stratification of cells in the ventral half of the AER. At later stages, this often leads to development of a secondary ventral AER, which can promote formation of an ectopic digit. The second AER forms at the juxtaposition of the ventral border of the broadened mutant AER and the distal border of an ectopic Lmx1b expression domain. Analysis of En1/Wnt7a double mutants demonstrates that the dorsalizing gene Wnt7a is required for the formation of the ectopic AERs in En1 mutants and for ectopic expression of Lmx1b in the ventral mesenchyme (Loomis, 1998).

En1 is not required for the process of AER induction or the initial stages of AER formation. Loss of En1 function has no apparent effect on the early thickening of the pre-AER ventral ectoderm or on the initial induction of AER marker genes, such as Dlx2 and FGF8. Cells of the dorsal AER of En1 mutants, like those of wild type limbs, do not express Wnt7a and they stratify and initiate Fgf4 expression, an AER differentiation marker. In contrast, differentiation of the ventral portion of the AER, where En1 is normally expressed, appears to be delayed and abnormal in En1 mutants. At a time when a mature AER is apparent in wild-type limbs, the ventral portion of the En1 mutant AER remains extended with the anterior region being much broader than the posterior. A model is suggested whereby, in En1 mutants, ectopic ventral Wnt7a and/or Lmx1b expression leads to the transformation of ventral cells in the broadened AER to a more dorsal phenotype. This leads to induction of a second zone of compaction ventrally, which in some cases goes on to form an autonomous secondary AER. It is proposed that in normal AER development, cells which give rise to the mouse AER initially overlie much of the presumptive limb mesoderm, and that sequential, convergent morphogenetic movements are required for normal ridge formation. A first wave of lateral morphogenetic movements results in the compaction of the AER precursor cells onto the ventral ectodermal thickening. A second wave compresses this domain to the distal 1/3 of the ventral limb, and a final wave constricts the cells into the densely packed AER. In En1 mutants the final wave is markedly inhibited and a secondary compaction process is initiated at the ventroproximal border of the widened mutant AER. The normal final wave of ectodermal movements resembles the closing of a zipper. This process initiates posteriorly and proceeds anteriorly, bringing the ventral domain of the wild-type AER into close proximity to the dorsal domain, which is anchored at the D-V interface. In En1 mutant limbs, the anatomically ventral AER cells take on dorsal characteristics, initiating a secondary AER (Loomis, 1998).

The gene Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb. R-fng is homologous to Drosophila fringe. R-fng is expressed in the dorsal ectoderm and apical ectodermal ridge (AER) prior to the expression of Fgf-8, a gene thought to play a role in the formation of the AER. Abnormal limb phenotypes consisting of AER-like structures are observed in 16% of embryos infected with a replication-competent retroviral vector containing R-fng, suggesting that R-fng misexpression perturbs the normal formation of the AER. While Wnt-7a, expressed in the dorsal ectoderm has no effect on R-fng expression, Engrailed-1 normally expressed in the ventral ectoderm, strongly represses R-fng and Wnt-7a, suggesting that En-1 regulates dorsal-ventral polarity of the limb and the positioning of the AER. This En-1 role contrasts with the role taken by Engrailed in Drosophila: the regulation of posterior compartment identity. It is suggested that the AER develops at the interface of tissue that is expressing high levels of R-fng adjacent to tissue that does not express R-fng. Because R-fng is not normally expressed on the ventral side of the limb, ectopic expression of R-fng here creates new boundaries that result in additional AERs. Serrate-2 (see Drosophila Serrate) is expressed in the AER from the earliest stages of its formation through at least stage 26. Chicken Notch-1 is also expressed in the AER. Thus R-fng, like its Drosophila counterpart, may act upstream of Notch signaling (Laufer, 1997 and Rodriguez-Esteban, 1997).

The Engrailed-1 gene, En1, a murine homolog of the Drosophila homeobox gene engrailed, is required for midbrain and cerebellum development and dorsal/ventral patterning of the limbs. In Drosophila, en is involved in regulating a number of key patterning processes including segmentation of the epidermis. During evolution, have the biochemical properties of En proteins been conserved? If so, this would reveal a common underlying molecular mechanism to their diverse developmental activities. To address this question, the coding sequences of En1 were replaced with Drosophila en. Mice expressing Drosophila en in place of En1 have a near complete rescue of the lethal En1 mutant brain defect and most skeletal abnormalities. In contrast, expression of Drosophila en in the embryonic limbs of En1 mutants does not lead to repression of Wnt7a in the embryonic ventral ectoderm or to full rescue of the embryonic dorsal/ventral patterning defects. Furthermore, neither murine En2 nor Drosophila en rescue the postnatal limb abnormalities that develop in the rare En1 null mutants that do survive. These studies demonstrate that the biochemical activity utilized in mouse to mediate brain development has been retained by Engrailed proteins across the phyla, and indicate that during evolution vertebrate En proteins have acquired two unique functions during embryonic and postnatal limb development and that only En1 can function postnatally (Hanks, 1998).

The limbs of En1 mutant animals expressing knock in-En2 appear normal at birth: En2 can repress embryonic expression of Wnt7a and Lmx1b in the ventral limb. However, by about 3-4 weeks post partum in En1 mutants with knock in-En2 and En1 mutants with knock in-Drosophila engrailed, the ventral epidermis overlying the palm pads becomes pigmented and begins to form nail-like structures. These postnatal defects correlate with postnatal expression of En1 in epidermal structures. Although it is possible that the postnatal limb defects in En1 mutants result from a lack of En1 during early embryonic limb patterning, it seems more likely that they are due to a requirement for En1 later in epidermal development. The fact that En2 can rescue the early dorsal/ventral patterning function of En1 in the limb but not a later function in the epidermis, and that Drosophila en cannot fully rescue either limb function of En1, suggests that the functions of En1 protein are different in early and late limb development. Furthermore, since both En2 and en can rescue the function of En1 in the brain, the region(s) of En1 required for this function must be conserved in both en and En2, unlike the region(s) required for limb development. It is speculated that the functional differences between the three engrailed proteins may reflect the inability of en and En2 to interact with the full repertoire of En1 accessory proteins, possibly resulting in altered DNA binding affinities for selective targets. Comparison of the sequences of all En proteins has identified five conserved domains, the largest being the homeodomain. Outside these domains En1 and En2, or En, diverge to a similar degree. Thus, in a non-conserved En region, the coding sequence of the first vertebrate En gene could have evolved a domain required for specific protein-protein or protein-DNA interactions in the limbs, which became further specialized in En1 after the second En gene (now designated En2) was formed by duplication. In this regard it is interesting to note that En1 proteins share a number of conserved regions that are not found in other En proteins. It is also possible that some of the conserved domains do not have the same repertoire of functions. It has been noted that EH-1, which is present in a number of transcription factors, can be divided into subfamilies based on sequence conservation. Furthermore, EH-1 is quite divergent between mouse and Drosophila. Although it was shown in a fly in vivo assay that EH-1 from mouse En1 can functionally replace EH-1 from En, it is possible that during mouse development Drosophila's En EH-1 can functionally replace En1 in the brain and skeleton, but not the limb. Thus, protein sequence and experimental data are consistent with the hypothesis that En1 protein has evolved one or more functional domains required for vertebrate limb development (Hanks, 1998).

Dorsoventral (DV) patterning of the vertebrate limb requires the function of the transcription factor Engrailed 1 (EN1) in the ventral ectoderm. EN1 restricts, to the dorsal half of the limb, the expression of the two genes known to specify dorsal pattern. Limb growth along the proximodistal (PD) axis is controlled by the apical ectodermal ridge (AER), a specialized epithelium that forms at the distal junction between dorsal and ventral ectoderm. Using retroviral-mediated misexpression of the bone morphogenetic protein (BMP) antagonist Noggin or an activated form of the BMP receptor in the chick limb, it has been demonstrated that BMP plays a key role in both DV patterning and AER induction. Thus, the DV and PD axes are linked by a common signal. Loss and gain of BMP function experiments show that BMP signaling is both necessary and sufficient to regulate EN1 expression, and consequently DV patterning. These results also indicate that BMPs are required during induction of the AER. Manipulation of BMP signaling results in either disruptions in the endogenous AER, leading to absent or severely truncated limbs or the formation of ectopic AERs that can direct outgrowth. Moreover, BMP controls the expression of the MSX transcription factors, and the results suggest that MSX acts downstream of BMP in AER induction. It is proposed that the BMP signal bifurcates at the level of EN1 and MSX to mediate differentially DV patterning and AER induction, respectively (Pizette, 2001).

The epithelial b variant of Fgfr2 is active in the entire surface ectoderm of the early embryo, and later in the limb ectoderm and AER, where it is required for limb outgrowth. Since limb buds do not form in the absence of Fgfr2, chimera analysis was used to investigate the mechanism of action of this receptor in limb development. ES cells homozygous for a loss-of-function mutation of Fgfr2 that carry a ß-galactosidase reporter were aggregated with normal pre-implantation embryos. Chimeras with a high proportion of mutant cells do not form limbs, whereas those with a moderate proportion form limb buds with a lobular structure and a discontinuous AER. Where present, the AER do not contain mutant cells, although mutant cells localize to the adjacent surface ectoderm and limb mesenchyme. In the underlying mesenchyme of AER-free areas, cell proliferation is reduced, and transcription of Shh and Msx1 is diminished. En1 expression in the ventral ectoderm is discontinuous and exhibits ectopic dorsal localization, whereas Wnt7a expression is diminished in the dorsal ectoderm but remains confined to that site. En1 and Wnt7a are not expressed in non-chimeric Fgfr2-null mutant embryos, revealing that they are downstream of Fgfr2. In late gestation chimeras, defects presented in all three limb segments as bone duplications, bone loss or ectopic outgrowths. It is suggested that Fgfr2 is required for AER differentiation, as well as for En1 and Wnt7a expression. This receptor also mediates signals from the limb mesenchyme to the limb ectoderm throughout limb development, affecting the position and morphogenesis of precursor cells in the dorsal and ventral limb ectoderm, and AER (Gorivodsky, 2003).

Mouse Engrailed: Function in muscle development

The molecular basis underlying the establishment of the myogenic lineage, subsequent differentiation, and the establishment of specific fiber types (i.e., fast versus slow) is becoming well understood. In contrast, the regulation of the general properties of a specific anatomical muscle group (e.g., leg versus jaw muscles) and the regulation of muscle-fiber properties within a particular group are less well characterized. The potential role played by murine Engrailed-2 (En-2), which is specifically expressed in myoblasts in the first arch and maintained in the muscles of mastication in the adult, has been investigated. Mice were generated that ectopically express En-2 in all muscles during early development and primarily in fast muscles in the adult. Ectopic En-2 in nonjaw muscles leads to a decrease in fiber size, whereas overexpression in the jaw muscles leads to a shift in fiber metabolic properties as well as a decrease in fiber size. In contrast, loss of En-2 in the jaw leads to a shift in fiber metabolic properties in the jaw of female mice only. Jaw muscles are sexually dimorphic, and it is proposed that the function of En-2 and mechanisms guiding sexual dimorphism of the jaw muscles are integrated. It is concluded that the specific expression of En-2 in the jaw therefore plays a role in specifying muscle-fiber characteristics that contribute to the physiologic properties of specific muscle groups (Degenhardt, 2001).

En-2 is expressed in mandibular arch myoblasts of the mouse. The activity of the En-2 enhancer is maintained in several functionally related muscles that arise from the first arch. Through the use of reporter transgenics, it has been demonstrated that local cell-cell interactions are important in maintaining En-2 expression in the mandibular arch cells. En-2 enhancer activity in the first arch requires a combination of cis-acting sequences that includes a motif that is identical to one found in the Otx2 enhancer, one which is sufficient to direct expression in the first arch. These data support the notion that cranial muscle development is regulated by local cell-cell interactions that distinguish distinct anatomical and functional muscle groups (Degenhardt, 2002).

Table of contents

The Interactive Fly resides on the
Society for Developmental Biology's Web server.