Placental failure in mice lacking the homeobox gene Dlx3.

Dlx3 is a homeodomain transcription factor and a member of the vertebrate Distal-less family. Targeted deletion of the mouse Dlx3 gene results in embryonic death between day 9.5 and day 10 because of placental defects that alter the development of the labyrinthine layer. In situ hybridization reveals that the Dlx3 gene is initially expressed in ectoplacental cone cells and chorionic plate, and later in the labyrinthine trophoblast of the chorioallantoic placenta, where major defects are observed in the Dlx3 -/- embryos. The expression of structural genes, such as 4311 and PL-1, which were used as markers to follow the fate of different derivatives of the placenta, was not affected in the Dlx3-null embryos. However, by day 10.5 of development, expression of the paired-like homeodomain gene Esx1 was strongly down-regulated in affected placenta tissue, suggesting that Dlx3 is required for the maintenance of Esx1 expression, normal placental morphogenesis, and embryonic survival.

Formation of Rathke's pouch requires dual induction from the diencephalon.

Targeted disruption of the homeobox gene T/ebp (Nkx2.1, Ttf1, Titf1) in mice results in ablation of the pituitary. Paradoxically, while T/ebp is expressed in the ventral diencephalon during forebrain formation, it is not expressed in Rathke's pouch or in the pituitary gland at any time of embryogenesis. Examination of pituitary development in the T/ebp homozygous null mutant embryos revealed that a pouch rudiment is initially formed but is eliminated by programmed cell death before formation of a definitive pouch. In the diencephalon of the mutant, Bmp4 expression is maintained, whereas Fgf8 expression is not detectable. These data and additional genetic and molecular observations suggest that Rathke's pouch develops in a two-step process that requires at least two sequential inductive signals from the diencephalon. First, BMP4 is required for induction and formation of the pouch rudiment, a role confirmed by analysis of Bmp4 homozygous null mutant embryos. Second, FGF8 is necessary for activation of the key regulatory gene Lhx3 and subsequent development of the pouch rudiment into a definitive pouch. This study provides firm molecular genetic evidence that morphogenesis of the pituitary primordium is induced in vivo by signals from the adjacent diencephalon.

Mediolateral patterning of somites: multiple axial signals, including Sonic hedgehog, regulate Nkx-3.1 expression.

The axial structures, the notochord and the neural tube, play an essential role in the dorsoventral patterning of somites and in the differentiation of their many cell lineages. Here, we investigated the role of the axial structures in the mediolateral patterning of the somite by using a newly identified murine homeobox gene, Nkx-3.1, as a medial somitic marker in explant in vitro assays. Nkx-3.1 is dynamically expressed during somitogenesis only in the youngest, most newly-formed somites at the caudal end of the embryo. We found that the expression of Nkx-3.1 in pre-somitic tissue explants is induced by the notochord and maintained in newly-differentiated somites by the notochord and both ventral and dorsal parts of the neural tube. We showed that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures by exposing explants to either COS cells transfected with a Shh expression construct or to recombinant SHH. Shh could induce and maintain Nkx-3.1 expression in pre-somitic mesoderm and young somites but not in more mature, differentiated ones. The effects of Shh on Nkr-3.1 expression were antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. Additionally, we confirmed that the expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial structures but that Shh by itself is not capable of inducing or maintaining it. We suggest that the establishment of somitic medial and lateral compartments and the early events in myogenesis are governed by a combination of positive and inhibitory signals derived from the neighboring structures, as has previously been proposed for the dorsoventral patterning of somites.

Multistep control of pituitary organogenesis.

Lhx3 and Lhx4 (Gsh4), two closely related LIM homeobox genes, determine formation of the pituitary gland in mice. Rathke's pouch is formed in two steps-first as a rudiment and later as a definitive pouch. Lhx3 and Lhx4 have redundant control over formation of the definitive pouch. Lhx3 controls a subsequent step of pituitary fate commitment. Thereafter, Lhx3 and Lhx4 together regulate proliferation and differentiation of pituitary-specific cell lineages. Thus, Lhx3 and Lhx4 dictate pituitary organ identity by controlling developmental decisions at multiple stages of organogenesis.

Hoxd-12 differentially affects preaxial and postaxial chondrogenic branches in the limb and regulates Sonic hedgehog in a positive feedback loop.

Several 5' members of the Hoxd cluster are expressed in nested posterior-distal domains of the limb bud suggesting a role in regulating anteroposterior pattern of skeletal elements. While loss-of-function mutants have demonstrated a regulatory role for these genes in the developing limb, extensive functional overlaps between various different Hox genes has hampered elucidation of the roles played by individual members. In particular, the function of Hoxd-12 in the limb remains obscure. Using a gain-of-function approach, we find that Hoxd-12 misexpression in transgenic mice produces apparent transformations of anterior digits to posterior morphology and digit duplications, while associated tibial hemimelia and other changes indicate that formation/growth of certain skeletal elements is selectively inhibited. If the digital arch represents an anterior bending of the main limb axis, then the results are all reconcilable with a model in which Hoxd-12 promotes formation of postaxial chondrogenic condensations branching from this main axis (including the anteriormost digit) and selectively antagonizes formation of 'true' preaxial condensations that branch from this main axis (such as the tibia). Hoxd-12 misexpression can also induce ectopic Sonic hedgehog (Shh) expression, resulting in mirror-image polydactyly in the limb. Misexpression of Hoxd-12 in other lateral plate derivatives (sternum, pelvis) likewise phenocopies several luxoid/luxate class mouse mutants that all share ectopic Shh signalling. This suggests that feedback activation of Shh expression may be a major function of Hoxd-12. Hoxd-12 can bind to and transactivate the Shh promoter in vitro. Furthermore, expression of either exogenous Hoxd-11 or Hoxd-12 in cultured limb bud cells, together with FGF, induces expression of the endogenous Shh gene. Together these results suggest that certain 5' Hoxd genes directly amplify the posterior Shh polarizing signal in a reinforcing positive feedback loop during limb bud outgrowth.

The Rx homeobox gene is essential for vertebrate eye development.

Development of the vertebrate eye requires a series of steps including specification of the anterior neural plate, evagination of the optic vesicles from the ventral forebrain, and the cellular differentiation of the lens and retina. Homeobox-containing genes, especially the transcription regulator Pax6, play a critical role in vertebrate and invertebrate eye formation. Mutations in Pax6 function result in eye malformations known as Aniridia in humans and Small eye syndrome in mice. The Drosophila homologue of Pax6, eyeless, is also necessary for correct invertebrate eye development, and its misexpression leads to formation of ectopic eyes in Drosophila. Here we show that a conserved vertebrate homeobox gene, Rx, is essential for normal eye development, and that its misexpression has profound effects on eye morphology. Xenopus embryos injected with synthetic Rx RNA develop ectopic retinal tissue and display hyperproliferation in the neuroretina. Mouse embryos carrying a null allele of this gene do not form optic cups and so do not develop eyes. The Rx gene family plays an important role in the establishment and/or proliferation of retinal progenitor cells.

Hypogonadism and obesity in mice with a targeted deletion of the Nhlh2 gene.

The family of basic helix-loop-helix (bHLH) genes comprises transcription factors involved in many aspects of growth and development. We have previously described two bHLH transcription factors, Nhlh1 and Nhlh2 (originally named NSCL1 and NSCL2). The nucleotide and predicted protein sequences of Nhlh1 and Nhlh2 are homologous within their bHLH domain where there are only three conservative amino acid differences. During murine embryogenesis, Nhlh1 and Nhlh2 share an overlapping but distinct pattern of expression in the developing nervous system. To improve our understanding of the role of these genes during neurogenesis, we have generated mice containing targeted deletions of both genes and here describe our results for Nhlh2. Loss of Nhlh2 results in a disruption of the hypothalamic-pituitary axis in mice. Male Nhlh2-/- mice are microphallic, hypogonadal and infertile with alterations in circulating gonadotropins, a defect in spermatogenesis and a loss of instinctual male sexual behaviour. Female Nhlh2-/- mice reared alone are hypogonadal, but when reared in the presence of males, their ovaries and uteri develop normally and they are fertile. Both male and female homozygotes exhibit progressive adult-onset obesity. Nhlh2 is expressed in the ventral-medial and lateral hypothalamus, Rathke's pouch and in the anterior lobe of the adult pituitary. Our results support a role for Nhlh2 in the onset of puberty and the regulation of body weight metabolism.

The Ames dwarf gene, df, is required early in pituitary ontogeny for the extinction of Rpx transcription and initiation of lineage-specific cell proliferation.

Two nonallelic dwarfing mutations in mice define genes important for pituitary development and function. Mice homozygous for either the Ames (df) or Snell (Pit 1dw) dwarf mutations exhibit severe proportional dwarfism, hypothyroidism, and infertility due to the cytodifferentiation failure of three anterior pituitary cell types: thyrotropes, somatotropes, and lactotropes. Analysis of double heterozygotes and double mutants has provided evidence that the df and dw genes act sequentially in the same genetic pathway. Double heterozygotes had no reduction in growth rate or final adult size. Double homozygotes had essentially the same phenotype as the single mutants and were recovered at the predicted frequency, indicating that there are no previously unrecognized, redundant functions of the two genes. Several lines of evidence demonstrate that df acts earlier in the differentiation pathway than Pit1. The df mutants fail to extinguish expression of the homeobox gene Rpx on embryonic day 13.5 (e13.5), and the size of their nascent pituitary glands is reduced by e14.5. In contrast, Pit1dw mutants down-regulate Rpx appropriately and exhibit normal cell proliferation up to e14.5. The failure to extinguish Rpx and the concomitant hypocellularity of df pituitaries suggest the importance of Rpx repression in lineage-specific cell proliferation before the appearance of lineage-specific markers. Later, Pit-1 and hypothalamic neuropeptides act sequentially to regulate marker gene transcription and cell proliferation. These results establish the time of df action in a cascade of genes that regulate pituitary ontogeny.

Specification of pituitary cell lineages by the LIM homeobox gene Lhx3.

During pituitary organogenesis, the progressive differentiation of distinct pituitary-specific cell lineages from a common primordium involves a series of developmental decisions and inductive interactions. Targeted gene disruption in mice showed that Lhx3, a LIM homeobox gene expressed in the pituitary throughout development, is essential for differentiation and proliferation of pituitary cell lineages. In mice homozygous for the Lhx3 mutation, Rathke's pouch formed but failed to grow and differentiate; such mice lacked both the anterior and intermediate lobes of the pituitary. The determination of all pituitary cell lineages, except the corticotrophs, was affected, suggesting that a distinct, Lhx3-independent ontogenetic pathway exists for the initial specification of this lineage.

Embryonic expression of glial cell-line derived neurotrophic factor (GDNF) suggests multiple developmental roles in neural differentiation and epithelial-mesenchymal interactions.

We describe the cloning of the mouse glial cell line-derived neurotrophic factor (GDNF) gene and its expression during embryogenesis. GDNF is a distant member of the superfamily of TGF-beta related genes that was originally identified on the basis of its striking neurotrophic activity. GDNF is expressed in a highly dynamic pattern in the anterior neuroectoderm during early stages of neurogenesis between E7.5 and E10.5. Beginning at E10.5 GDNF is also expressed in several organs that develop through inductive epithelial-mesenchymal interactions. In those organs, GDNF expression is strictly confined to mesenchymal tissues and is not found in epithelia. Our results suggest multiple roles for GDNF during early stages of neuronal development and in epithelial-mesenchymal interactions.

Rpx: a novel anterior-restricted homeobox gene progressively activated in the prechordal plate, anterior neural plate and Rathke's pouch of the mouse embryo.

We have isolated a new murine homeobox gene, Rpx (for Rathke's pouch homeobox), that is dynamically expressed in the prospective cephalic region of the embryo during gastrulation. Early expression is seen in the anterior midline endoderm and prechordal plate precursor. Expression is subsequently activated in the overlying ectoderm of the cephalic neural plate, suggesting that inductive contact with Rpx-expressing mesendoderm is required for this expression. Subsequently, Rpx expression is extinguished in the mesendoderm while remaining in the prospective prosencephalic region of the neural plate ectoderm. Ultimately, transcripts become restricted to Rathke's pouch, the primordium of the pituitary, which is known to be derived from the most anterior ectoderm of the early embryo. Down regulation of Rpx in the pouch coincides with the differentiation of pituitary-specific cell types. Rpx is the earliest known marker for the pituitary primordium, suggestive of a role in the early determination or differentiation of the pituitary. Since Rpx is expressed so dynamically and so early in the anterior region of the embryo, and since its early expression domain is much more extensive than the region fated to form the pituitary, it is likely that Rpx is involved in the initial determination of the anterior (prechordal) region of the embryo.

Gnot1, a member of a new homeobox gene subfamily, is expressed in a dynamic, region-specific domain along the proximodistal axis of the developing limb.

Limb development endures as an excellent model for pattern formation in vertebrates. We have identified Gnot1 as a member of a new homeobox gene subfamily. Gnot1 is expressed in a dynamic temporospatial distribution in the developing limb, initially correlating with regions destined to form distal structures and then becoming progressively more restricted to specific regions determined to give rise to wrist and ankle. Micro-surgical alteration of the developmental program of the limb reveals that Gnot1 is expressed in a position- and fate-dependent manner, responding both to signals from the apical ridge and the polarizing zone. Furthermore, Gnot1 activation by polarizing signals occurs temporally downstream of Hoxd gene activation, but well before the first appearance of condensations that will give rise to the carpus of the wrist. The features of Gnot1 expression suggest a role for this gene in regulating pattern formation during limb development.

A Xenopus distal-less gene in transgenic mice: conserved regulation in distal limb epidermis and other sites of epithelial-mesenchymal interaction.

In this paper, we show the conserved regulation of the homeodomain gene Distal-less-3 (Dlx-3) by analyzing the expression of a promoter from the Xenopus ortholog, Xdll-2, in transgenic mice. A 470-bp frog regulatory sequence confers appropriate expression on a lacZ reporter gene in the ectodermal component of structures derived from epithelial-mesenchymal interactions. Remarkably, this includes structures absent in Xenopus, such as the hair follicle and mammary gland, suggesting that conserved regulatory elements can be used to control the formation of structures peculiar to individual species. In addition, expression of Dlx-3 in developing limbs is highest at the most distal portion. This pattern is duplicated by the Xenopus promoter, indicating that this DNA may include sequences responsive to conserved proximodistal patterning signals in the vertebrate limb.

Contrasting roles for c-Myc and L-Myc in the regulation of cellular growth and differentiation in vivo.

Although myc family genes are differentially expressed during development, their expression frequently overlaps, suggesting that they may serve both distinct and common biological functions. In addition, alterations in their expression occur at major developmental transitions in many cell lineages. For example, during mouse lens maturation, the growth arrest and differentiation of epithelial cells into lens fiber cells is associated with a decrease in L- and c-myc expression and a reciprocal rise in N-myc levels. To determine whether the down-regulation of L- and c-myc are required for mitotic arrest and/or completion of differentiation and whether these genes have distinct or similar activities in the same cell type, we have studied the consequences of forced L- and c-myc expression in the lens fiber cell compartment using the alpha A-crystallin promoter in transgenic mice (alpha A/L-myc and alpha A/c-myc mice). With respect to morphological and molecular differentiation, alpha A/L-myc lenses were characterized by a severely disorganized lens fiber cell compartment and a significant decrease in the expression of a late-stage differentiation marker (MIP26); in contrast, differentiation appeared to be unaffected in alpha A/c-myc mice. Furthermore, an analysis of proliferation indicated that while alpha A/L-myc fiber cells withdrew properly from the cell cycle, inappropriate cell cycle progression occurred in the lens fiber cell compartment of alpha A/c-myc mice. These observations indicate that continued late-stage expression of L-myc affected differentiation processes directly, rather than indirectly through deregulated growth control, whereas constitutive c-myc expression inhibited proliferative arrest, but did not appear to disturb differentiation. As a direct corollary, our data indicate that L-Myc and c-Myc are involved in distinct physiological processes in the same cell type.

Differential and overlapping expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less homeobox genes in craniofacial development.

During the development of the vertebrate head, cranial neural crest cells migrate into the branchial arches to form many of the structures of the facial skeleton. These cells follow defined developmental pathways and their fates are determined early. We have isolated and characterized the murine Distal-less homeobox gene Dlx-3 and have performed a comparative analysis of Dlx-3 and Dlx-2 expression during craniofacial development. In contrast to Dlx-2 and other vertebrate Distal-less genes, Dlx-3 is not expressed in the central nervous system and is expressed in a highly restricted region of the branchial arches. Dlx-2 and -3 display temporal and spatial differences in expression in the arches and their derivatives. In later development, these two genes are expressed in both complementary and partially overlapping domains in regions whose development is dependent on epithelial-mesenchymal interactions, such as the developing middle and inner ear, teeth and whisker follicles. The differential expression of Dlx genes in the branchial region suggests that they play key roles in craniofacial patterning and morphogenesis.

Detection of genes that are differentially expressed during mouse embryogenesis by genetic trapping strategies.

OBJECTIVE: Our objective was to identify novel genes that are expressed in temporally and spatially restricted patterns during mouse embryonic development and organogenesis. STUDY DESIGN: Two genetic trapping reporter constructs that lack transcriptional regulatory sequences were introduced independently into transcriptionally active gene loci by electroporation into mouse embryonic stem cells. Patterns of host gene-reporter construct expression were investigated in differentiated embryonic stem cells, embryoid bodies, and chimeric embryos at various stages of development. RESULTS: Three patterns of host gene-reporter construct expression were observed from the developmental analysis of four vector-integrated cell lines. Reporter expression patterns reflecting developmental regulation, constitutive activity, and developmental inactivation of the host genes were observed. CONCLUSIONS: Two vector-integrated gene loci from cell lines CCE-1C1 and D3-B44 have expression patterns not previously described by genetic trapping. Molecular characterization of these interrupted genes will shed light on their developmental function.

Development shows some backbone. HFSP Workshop on Genetic Control of Vertebrate Development cosponsored by the Human Frontier Science Program, European Science Foundation, and European Molecular Biology Organization, Les Diablerets, Switzerland, May 26-30, 1991.

This meeting aptly illustrated the power of a combined analysis of development in a range of vertebrate systems. Each system has its own inherent strengths: the mouse has gene transfer technology and targeted mutagenesis, the frog and chick have experimental embryology, and the zebrafish has genetics. It is the synergistic effect of considering all of these systems in combination that is without measure. In the past, the study of vertebrate development has been relegated to a largely descriptive phase. Initially, this was through analysis of morphological changes taking place during development. More recently, this has taken the form of cataloging the expression patterns of genes transcribed in development. It is clear that we are now entering an era when a functional analysis of development can get underway.

Molecular characterization of NSCL, a gene encoding a helix-loop-helix protein expressed in the developing nervous system.

We report here the molecular cloning and chromosomal localization of an additional member of the helix-loop-helix (HLH) family of transcription factors, NSCL. The NSCL gene was identified based on its hybridization to the previously described hemopoietic HLH gene, SCL. Murine NSCL cDNA clones were obtained from a day 11.5 mouse embryo cDNA library. The coding region is 399 base pairs and encodes a predicted protein of 14.8 kDa. The nucleotide sequence shows 71% identity and the amino acid sequence shows 61% identity to murine SCL in the HLH domain. The NSCL protein-coding region terminates six amino acids beyond the second amphipathic helix of the HLH domain. Expression of NSCL was detected in RNA from mouse embryos between 9.5 and 14.5 days postcoitus, with maximum levels of expression at 10.5-12 days. Examination of 12- and 13-day mouse embryos by in situ hybridization revealed expression of NSCL in the developing nervous system. The NSCL gene was mapped to murine chromosome 1. The very restricted pattern of NSCL expression suggests an important role for this HLH protein in neurological development.

Spatially restricted expression of a member of a new family of murine Distal-less homeobox genes in the developing forebrain.

The homeobox genes of Drosophila perform key functions in embryonic pattern formation, and their vertebrate counterparts may play similar developmental roles. Using polymerase chain reaction technology, we have identified four murine homologs of the Drosophila Distal-less homeobox gene that are expressed in midgestation embryos. The homeodomains encoded by these genes vary considerably from other known homeodomain sequences and represent a new family of vertebrate homeobox genes. We isolated a cDNA for one of these genes (Dlx-2) and studied its expression by in situ hybridization from 8.5 days postcoitum (pc) until postnatal day 1. Dlx-2 shows a restricted pattern of expression in the ventral forebrain, extending from the olfactory bulb to the ventral diencephalon. This domain of expression may delineate an ontogenetically defined subdivision within the forebrain. The murine Distal-less genes are the first homeobox genes described whose expression in the central nervous system is exclusively restricted to the forebrain. Thus, the Distal-less genes may contribute missing positional cues not provided by the previously identified vertebrate homeobox genes.