Kathleen A. Mahon

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, Rathkes 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.

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.

Expression and activity of L-Myc in normal mouse development.

To determine the role of L-Myc in normal mammalian development and its functional relationship to other members of the Myc family, we determined the normal patterns of L-myc gene expression in the developing mouse by RNA in situ hybridization and assessed the phenotypic impact of L-Myc deficiency produced through standard gene targeting methodology. L-myc transcripts were detected in the developing kidney and lung as well as in both the proliferative and the differentiative zones of the brain and neural tube. Despite significant expression of L-myc in developing mouse tissue, homozygous null L-myc mice were found to be viable, reproductively competent, and represented in expected frequencies from heterozygous matings. A detailed histological survey of embryonic and adult tissues, characterization of an embryonic neuronal marker, and measurement of cellular proliferation in situ did not reveal any congenital abnormalities. The lack of an apparent phenotype associated with L-Myc deficiency indicates that L-Myc is dispensable for gross morphological development and argues against a unique role for L-Myc in early central nervous system development as had been previously suggested. Although overlapping expression patterns among myc family members raise the possibility of complementation of L-Myc deficiency by other Myc oncoproteins, compensatory changes in the levels of c- and/or N-myc transcripts were not detected in homozygous null L-myc mice.

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.

A novel inducible element, activated by contact with Rathke's pouch, is present in the regulatory region of the Rpx/Hesx1 homeobox gene.

Reciprocal inductive interactions are postulated to play a role in the determination and differentiation of the pituitary gland and the ventral hypothalamus. The homeobox gene Rpx/Hesxl is expressed during gastrulation in the anterior endoderm, prechordal plate, and the prospective cephalic neural plate, and at later stages of development in Rathkes pouch, the primordium of the pituitary. We have defined the regulatory elements necessary for proper spatial and temporal expression during development in transgenic mice using lacZ reporter genes. Proper spatial and temporal expression in the anterior endoderm prechordal plate and anterior neural plate can be recapitulated with as little as 568 bp of upstream sequence and intragenic sequence containing the first exon and intron. Late-stage expression in Rathkes pouch requires additional negative and positive regulatory elements. Interestingly, deletion analysis uncovered an element that directs transgene expression to a region of the hypothalamus that lies in direct contact with Rathkes pouch. In vitro tissue recombination experiments have established that this expression is induced by contact with the pouch. We propose that this element may be present in other genes that normally respond to signals emanating from the pouch during the development of the hypothalamic-pituitary axis. The Rpx-lacZ transgenic mice provide a novel model system for the molecular dissection of inductive cell signaling during pituitary development.

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.

Chapter 24. The Role of Homeobox Genes in Vertebrate Embryonic Development

Publisher Summary This chapter discusses the role of homeobox genes in vertebrate embryonic development. The first vertebrate homeobox containing gene to be identified was isolated from xenopus. Subsequently, a large number of genes were isolated from three vertebrate species—xenopus, mouse, and humans. Approximately 40 mammalian genes have been isolated that contain homeobox sequences most similar to that of the Drosophila antp gene. These genes are collectively known as Hox genes and represent the largest group of vertebrate homeobox genes known to date. Many Hox gene family members are differentially expressed in the vertebrate limb bud, often with a graded pattern of expression. In xenopus, functional aspects of homeobox gene activity have been studied by ectopic expression or overexpression of these genes or by ablation of their gene products in embryos. Xenopus homeobox genes have been shown to be involved in pattern formation. Gain-of-function phenotypes have been similarly generated in mice, with ectopic expression and overexpression of homeobox gene constructs being achieved through the use of transgenic technology. Loss-of-function phenotypes can be obtained through the use of embryonic stem (ES) cells, in which genes can be mutated via homologous recombination. These gain of function and loss-of-function studies clearly illustrate the developmental importance of homeobox gene activity for proper vertebrate morphogenesis. There are, however, several naturally occurring mutations that have been identified in non- antp class homeobox genes. At least two known mouse mutations affecting morphogenesis have been shown to bear mutations in homeobox genes. There is also convincing evidence that aberrant homeobox gene expression can contribute to the genesis or progression of cancer. These data provide intriguing clues concerning the developmental role of the homeobox genes in vertebrates. It is likely that the further analysis of homeobox genes can implicate them in human disease and developmental malformation.

Specificity of Gene Expression and Insertional Mutagenesis in Transgenic Mice

Transgenic mice constitute an ideal system for determining the spatial and temporal control of gene expression in every conceivable tissue of the mammalian organism. A wide variety of gene constructs have been inserted in the mouse germ line, and the initial data compiled with this system have recently been reviewed by Gordon and Ruddle (1985) and by Palmiter and Brinster (1985). One of the most important conclusions from this initial work has been that expression of transferred genes may be directed to specific organs and tissues of the transgenic mouse, irrespective of the site of integration in the genome. In addition, a number of chimeric genes containing a 5′ flanking region of gene A and the coding sequence of gene B were found to express gene product B in the target tissue determined by gene A. Examples from our own laboratories that corroborate this fact include chimeric gene constructs composed of untranslated control regions of the mouse αA crystallin gene, mouse α2(I) collagen gene, or avian Rous sarcoma virus (RSV) and the bacterial sequence that encodes chloramphenicol acetyltransferase (CAT). In each instance, the upstream sequences exerted specific spatial and temporal controls on CAT expression in the transgenic mouse. A case in point is the αA crystallin control region that was found to produce the CAT enzyme selectively in the lens of the eye (Overbeek et al., 1985).