The prospect of using cell replacement therapies has raised the key issue of whether elucidation of developmental pathways can facilitate the generation of therapeutically important cell types from stem cells. Here we show that the homeodomain proteins Lmx1a and Msx1 function as determinants of midbrain dopamine neurons, cells that degenerate in patients with Parkinson's disease. Lmx1a is sufficient and required to trigger dopamine cell differentiation. An early activity of Lmx1a is to induce the expression of Msx1, which complements Lmx1a by inducing the proneural protein Ngn2 and neuronal differentiation. Importantly, expression of Lmx1a in embryonic stem cells results in a robust generation of dopamine neurons with a "correct" midbrain identity. These data establish that Lmx1a and Msx1 are critical intrinsic dopamine-neuron determinants in vivo and suggest that they may be essential tools in cell replacement strategies in Parkinson's disease.

Regeneration of appendages is frequent among invertebrates as well as some vertebrates. However, in mammals this has been largely relegated to digit tip regeneration, as found in mice and humans. The regenerated structures are formed from a mound of undifferentiated cells called a blastema, found just below the site of amputation. The blastema ultimately gives rise to all of the tissues in the regenerate, excluding the epidermis, and has classically been thought of as a homogenous pool of pluripotent stem cells derived by dedifferentiation of stump tissue, although this has never been directly tested in the context of mammalian digit tip regeneration. Successful digit tip regeneration requires that the level of amputation be within the nail bed and depends on expression of Msx1. Because Msx1 is strongly expressed in the nail bed mesenchyme, it has been proposed that the Msx1-expressing cells represent a pluripotent cell population for the regenerating digit. In this report, we show that Msx1 is dynamically expressed during digit tip regeneration, and it does not mark a pluripotent stem cell population. Moreover, we show that both the ectoderm and mesoderm contain fate-restricted progenitor populations that work in concert to regenerate their own lineages within the digit tip, supporting the hypothesis that the blastema is a heterogeneous pool of progenitor cells.

A new mouse Hox locus, Hox‐7, is defined on chromosome 5 by a gene homologous to the Drosophila gene msh, which contains a homeobox sequence distantly related to that of Antennapedia. By in situ hybridization, expression of Hox‐7 is detected in the neural fold of embryos, and also in cephalic neural crest. In addition, expression takes place in the developing valves of the embryonic heart. Mandibular and hyoid arches are strongly labelled, expression becoming restricted to the most distal part of mouth and face processes as development proceeds. Intense labelling is also observed in developing limb buds, in the distal region which has been shown to be essential for limb morphogenesis. The pronounced accumulation and regional localization of Hox‐7 transcripts in mandibular and limb processes point to a specific morphogenetic role for this mouse homeobox gene.

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We have generated a null allele of the mouse Msx1 homeobox gene by insertion of an nlacZ reporter gene into its homeobox. The sensitivity of beta-galactosidase detection permitted us to reveal novel aspects of Msx1 gene expression in heterozygous embryos, in particular in ectoderm and mesoderm during gastrulation, and in migrating neural crest cells. Homozygous mutant mice die at birth with facial defects (see Satokata, I. and Maas, R. (1994) Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat. Genet. 6, 348-356). To investigate the reason for this limited phenotype, we compared the pattern of Msx1 expression with that of the closely related Msx2 gene in wild type embryos and in Msx1-/- mutants. Notably, whereas the expression of Msx1 and Msx2 overlap in the developing limb, this is not the case in the facial regions most affected in the mutant.

The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and,subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.

Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA (Msx1-AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontoblastic cell line (MO6-G3) showed that the balance between the levels of the two Msx1 RNAs is related to the expression of Msx1 protein. To analyze the impact of this balance in the Msx-Dlx homeoprotein pathway, we analyzed the effect of Msx1, Msx2, and Dlx5 overexpression on proteins involved in skeletal differentiation. We showed that the Msx1-AS RNA is involved in crosstalk between the Msx-Dlx pathways because its expression was abolished by Dlx5. Msx1 was shown to down-regulate a master gene of skeletal cells differentiation, Cbfa1. All these data strongly suggest that the ratio between Msx1 sense and antisense RNAs is a very important factor in the control of skeletal terminal differentiation. Finally, the initiation site for Msx1-AS RNA transcription was located by primer extension in both mouse and human in an identical region, including a consensus TATA box, suggesting an evolutionary conservation of the AS RNA-mediated regulation of Msx1 gene expression. M sx genes are homeobox genes related to the Drosophila msh (muscle segment homeobox)-like gene family. Msx homeogenes play an important role in inductive epithelio-mesenchymal interactions leading to vertebrate organogenesis (1). Among this family, Msx1 is a fundamental factor for craniofacial skeleton formation. In mouse, head Msx1 gene expression is located mainly in regions of cephalic neural crest cell migration and differentiation, as well as in the derived mesenchymal cells (2-4). Msx1 also is found in a variety of embryonic tissues requiring epithelio-mesenchymal interactions for their morphogenesis such as limb bud, embryonic tail, hair follicle, and tooth bud.Msx1-deficient mice exhibit dental and craniofacial malformations, such as cleft palate, reduced mandible length, abnormalities of nasal, frontal, and parietal bones, as well as arrested tooth development, suggesting a role of Msx1 in outgrowth of these tissues (5, 6). In humans, mutations in Msx1 gene have been involved in tooth agenesis (7-9) and cleft palate (10), and the phenotype was proposed to be related to a dose effect of Msx1 protein (9). Interestingly, Msx1 down-regulation is associated with the terminal differentiation of several cell types such as cartilage (4,11,12) and muscle (13); indeed, in muscle cells, Msx1-forced expression results in a highly proliferative transformed phenotype and blocks myogenic terminal differentiation (14, 15) through the inhibition of a master gene expression, MyoD, by Msx1 (16). Thus, Msx1 is thought to prevent differentiation and enhance proliferation. Other...

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