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Although numerous molecules required for limb bud formation have recently been identified, the molecular pathways that initiate this process and ensure that limb formation occurs at specific axial positions have yet to be fully elucidated. Based on experiments in the chick, Fgf8 expression in the intermediate mesoderm (IM) has been proposed to play a critical role in the initiation of limb bud outgrowth via restriction of Fgt10 expression to the appropriate region of the lateral plate mesoderm. Contrary to the outcome predicted by this model, ablation of Fgf8 expression in the intermediate mesoderm before limb bud initiation had no effect on initial limb bud outgrowth or on the formation of normal limbs. When their expression patterns were first elucidated, both Fgf4 and Fgf8 were proposed to mediate critical functions of the apical ectodermal ridge (AER), which is required for proper limb bud outgrowth. Although mice lacking Fgf4 in the AER have normal limbs, limb development is severely affected in Fgf8 mutants and certain skeletal elements are not produced. By creating mice lacking both Fgf4 and Fgf8 function in the forelimb AER, we show that limb bud mesenchyme fails to survive in the absence of both FGF family members. Thus, Fgf4 is responsible for the partial compensation of distal limb development in the absence of Fgf8. A prolonged period of increased apoptosis, beginning at 10 days of gestation in a proximal-dorsal region of the limb bud, leads to the elimination of enough mesenchymal cells to preclude formation of distal limb structures. Expression of Shh and Fgf10 is nearly abolished in double mutant limb buds. By using a CRE driver expressed in both forelimb and hindlimb ectoderm to inactivate Fgf4 and Fgf8, we have produced mice lacking all limbs, allowing a direct comparison of FGF requirements in the two locations. Keywords: FGF; Limb development; Mouse; Intermediate mesoderm; Limb bud initiation: Apical ectodermal ridge; Cell survival; Apoptosis

To address the expression and function of Hoxb13, the 5' most Hox gene in the HoxB cluster, we have generated mice with loss-of-function and [beta]-galactosidase reporter insertion alleles of this gene. Mice homozygous for Hoxb13 loss-of-function mutations show overgrowth in all major structures derived from the tail bud, including the developing secondary neural tube (SNT), the caudal spinal ganglia, and the caudal vertebrae. Using the [beta]-galactosidase reporter allele of Hoxb13, also a loss-of-function allele, we found that the expression patterns of Hoxb13 in the developing spinal cord and caudal mesoderm are closely associated with overgrowth phenotypes in the tails of homozygous mutant animals. These phenotypes can be explained by the observed increased cell proliferation and decreased levels of apoptosis within the tail of homozygous mutant mice. This analysis of Hoxb13 function suggests that this 5' Hox gene may act as an inhibitor of neuronal cell proliferation, an activator of apoptotic pathways in the SNT, and as a general repressor of growth in the caudal vertebrae. Keywords: Hoxb genes; Hoxb13; Secondary neurulation; Axial skeleton; Tail; Apoptosis; Proliferation

The Hox genes encode a group of transcription factors essential for proper development of the mouse. Targeted mutation of the Hoxd11 gene causes reduced male fertility, vertebral transformation, carpal bone fusions, and reductions in digit length. A duplication of the Hoxd11 gene was created with the expectation that the consequences of restricted overexpression in the appropriate cells would provide further insight into the function of the Hoxd11 gene product. Genetic assays demonstrated that two tandem copies of Hoxd11 were functionally indistinguishable from the normal two copies of the gene on separate chromosomes with respect to formation of the axial and appendicular skeleton. Extra copies of Hoxd11 caused an increase in the lengths of some bones of the forelimb autopod and a decrease in the number of lumbar vertebrae. Further, analysis of the Hoxd11 duplication demonstrated that the Hoxd11 protein can perform some functions supplied by its paralogue Hoxa11. For example, the defects in forelimb bones are corrected when extra copies of Hoxd11 are present in the Hoxa11 homozygous mutant background. Thus, it appears that Hoxd11 can quantitatively compensate for the absence of Hoxa11 protein, and therefore Hoxa11 and Hoxd11 are functionally equivalent in the zeugopod. However, extra copies of Hoxd11 did not improve male or female fertility in Hoxa11 mutants. Interestingly, the insertion of an additional Hoxd11 locus into the HoxD complex does not appear to affect the expression patterns of the neighboring Hoxd10, -d12, or -d13 genes. Key Words: Hox genes; gene duplications; limb development; axial skeleton development; gene targeting.

The thymus, thyroid, and parathyroid glands in vertebrates develop from the pharyngeal region, with contributions both from pharyngeal endoderm and from neural crest cells in the pharyngeal arches. Hoxa3 mutant homozygotes have defects in the development of all three organs. Roles for the Hoxa3 paralogs, Hoxb3 and Hoxd3, were investigated by examining various mutant combinations. The thyroid defects seen in Hoxa3 single mutants are exacerbated in double mutants with either of its paralogs, although none of the double-mutant combinations resulted in thyroid agenesis. The results indicate that the primary role of these genes in thyroid development is their effect on the development and migration of the ultimobranchial bodies, which contribute the parafollicular or C-cells to the thyroid. Hoxb3,Hoxd3 double mutants show no obvious defects in the thymus or parathyroids. However, the removal of one functional copy of Hoxa3 from the Hoxb3,Hoxd3 double mutants ([Hoxa3.sup.+/-],[Hoxb3.sup.-/-],[Hoxd3.sup.-/-]) results in the failure of the thymus and parathyroid glands to migrate to their normal positions in the throat. Very little is known about the molecular mechanisms used to mediate the movement of tissues during development. These results indicate that Hoxa3, Hoxb3, and Hoxd3 have highly overlapping functions in mediating the migration of pharyngeal organ primordia. In addition, Hoxa3 has a unique function with respect to its paralogs in thymus, parathyroid, and thyroid development. This unique function may be conferred by the expression of Hoxa3, but not Hoxb3 nor Hoxd3, in the pharyngeal pouch endoderm.

Previous studies have shown that early mouse embryos with both FGF-4 alleles inactivated are developmentally arrested shortly after implantation. To understand the roles of FGF-4 during early development, we prepared genetically engineered embryonic stem (ES) cells, which are unable to produce FGF-4. Specifically, we describe the isolation and characterization of ES cells with both FGF-4 alleles inactivated. The FGF-4-/- ES cells do not require FGF-4 to proliferate in vitro, and addition of FGF-4 to the medium has little or no effect on their growth. Thus, FGF-4 does not appear to act as an autocrine growth factor for cultured ES cells. We also demonstrate that FGF-4-/- ES cells, like their unmodified counterparts, are capable of forming highly complex tumors in syngeneic mice composed of a wide range of differentiated cells types, including neural tissue, glandular epithelium, and muscle. In addition, we demonstrate that the FGF-4-/- ES cells can differentiate in vitro after exposure to retinoic acid; however, the growth and/or survival of the differentiated cells is severely compromised. Importantly, addition of FGF-4 to the culture medium dramatically increases the number of differentiated cells derived from the FGF-4-/- ES cells, in particular cells with many of the properties of parietal extraembryonic endoderm. Finally, we demonstrate that there are differences in the RNA profiles expressed by the differentiated progeny formed in vitro from FGF-4-/- ES cells and FGF-4+/+ ES cells when they are cultured with FGF-4. Taken together, the studies described in this report indicate that certain lineages formed in vitro are affected by the inactivation of the FGF-4 gene, in particular specific cells that form during the initial stage of ES cell differentiation. Thus, ES cells with both FGF-4 alleles inactivated should shed light on the important roles of FGF-4 during the early stages of mammalian development and help determine why FGF-4-/- embryos die shortly after implantation.

Hox genes encode transcription factors that are used to regionalize the mammalian embryo. Analysis of mice carrying targeted mutations in individual and multiple Hox genes is beginning to reveal a complex network of interactions among these closely related genes which is responsible for directing the formation of spatially restricted tissues and structures. In this report we present an analysis of the genetic interactions between all members of the third paralogous group, Hoxa3, Hoxb3, and Hoxd3. Previous analysis has shown that although mice homozygous for loss-of-function mutations in either Hoxa3 or Hoxd3 have no defects in common, mice mutant for both genes demonstrate that these two genes strongly interact in a dosage-dependent manner. To complete the analysis of this paralogous gene family, mice with a targeted disruption of the Hoxb3 gene were generated. Homozygous mutants have minor defects at low penetrance in the formation of both the cervical vertebrae and the IXth cranial nerve. Analysis and comparison of all double-mutant combinations demonstrate that all three members of this paralogous group interact synergistically to affect the development of both neuronal and mesenchymal neural crest-derived structures, as well as somitic mesoderm-derived structures. Surprisingly, with respect to the formation of the cervical vertebrae, mice doubly mutant for Hoxa3 and Hoxd3 or Hoxb3 and Hoxd3 show an indistinguishable defect, loss of the entire atlas. This suggests that the identity of the specific Hox genes that are functional in a given region may not be as critical as the total number of Hox genes operating in that region.

Mice were generated with a targeted disruption of the homeobox-containing gene hoxb-9. Mice homozygous for this mutation show defects in the development of the first and second ribs. In most cases the first and second ribs are fused near the point at which the first and second pairs of ribs normally attach to the sternum. Abnormalities of the sternum accompany the rib fusions. These include abnormal attachment of the ribs to the sternum, a reduction in the number of intercostal segments of the sternum, and abnormal growth of the intercostal segments. Over half of the homozygous mutants, as well as some heterozygotes, also have an eighth rib attached to the sternum. These results show that hoxb-9 plays a significant role in the specification of thoracic skeletal elements. To reveal potential interactions between the paralogous Hox genes hoxa-9 and hoxb-9, mice heterozygous for both mutations were intercrossed. Mice homozygous for both mutations show more severe phenotypes than predicted by the addition of the individual mutant phenotypes. Both the penetrance and the expressivity of the rib and sternal defects are increased, suggesting synergistic interactions between these genes. In particular, the sternum defects are greatly exacerbated. Interestingly, the defects in hoxb-9 and hoxa-9/ hoxb-9 mutant mice are concentrated along the axial column at points of transition between vertebral types.

Mice carrying a nonfunctional allele of hoxc-4 have been generated by gene targeting. The phenotype of mice homozygous for this mutation is strikingly different from those reported in mice lacking the paralogous genes hoxa-4, hoxb-4, and hoxd-4. In contrast to the mutants of the paralogous family members, hoxc-4 homozygotes do not manifest abnormalities in the cervical vertebrae, but instead show vertebral defects that extend from the second thoracic vertebra (t2) to t11. Therefore, defects do not correspond to the anterior limit of expression of hoxc-4, but rather begin within the region of strong hoxc-4 expression in the prevertebral anlagen (i.e., pv7-14). While hoxc-4 mutant homozygotes that reach adulthood are fertile and appear outwardly normal, most die before weaning age. The high lethality appears to result from partial or complete blockage of the lumen of the esophagus over a large portion of its length, as well as disorganization of the esophageal musculature. Although the Drosophila homolog of hoxc-4, Deformed, is autoregulated, mutation of the hoxc-4 gene does not affect transcription of its paralogous family members. However, in hoxc-4 mutant embryos, transcription of both the hoxc-5 and hoxc-6 genes is altered. Employment of cis/trans analysis showed that the hoxc-4 mutation acts in cis to affect the pattern of hoxc-5 expression. Therefore, this mutation is likely to cause a reduction of hoxc-5 function as well as complete loss of hoxc-4 function.

Mice possessing a null mutation at the Wnt3 locus fail to gastrulate and die at early stages of embryogenesis (E9.5). We have generated mice with a conditional ('fioxed') allele of Wnt3 (Wnt3c) in order to determine its roles at later stages of development. We have also generated transgenic mouse lines harboring Cre recombinase driven by the Brachyury (T) promoter as a means of specifcally removing Wnt3 in the primitive streak of embryos. We observed that mice transheterozygous for the Wnt3 null and conditional alleles (Wnt3n/c) and also possessing a 'TCre' line (Tcre14) exhibited no gastrulation anomalies but surprisingly possessed severe hindlimb outgrowth defects. We subsequently found that Tcre14 exhibited robust Cre activity in the hindlimb ectoderm. In addition, we found Wnt3 to be expressed ubiquitously in the ectoderm but not the mesenchyme of the developing limb. We demonstrate that these conditional mutants do not express markers characteristic of the apical ectodermal ridge (AER) suggesting that Wnt3 plays an important role in the establishment of this tissue. We also show that Fgf10, which is also essential for the establishment of the AER, continues to be strongly expressed in the early limb mesenchyme of these mutants suggesting that Wnt3 is likely to act downstream of this molecule. We describe further molecular characterization of the mutant hindlimbs. Finally, we describe the conditional removal of [Beta]-catenin in the limb mesenchyme vs ectoderm and argue that Wnt3 signals to the distal limb ectoderm to establish the AER.

In mice containing mutant alleles of either Hoxa11 or Hoxd11, kidney development appears grossly normal while animals carrying the combined, double mutations display hypoplastic defects in the newborn kidney. Based on many observations of Hox gene functional redundancy and the apparent functional overlap of Hoxa11 and Hoxd11 in kidney development, mice were created in which all three Hox11 paralogs, Hoxa11, Hoxc11, and Hoxd11, were introduced into a single animal. Combination of all three mutant alleles results in a complete loss of ureteric bud formation, one of the earliest steps in metanephric development. By E10.5, aggregation of the metanephric blastema and expression of Pax2 and WT1, two of the earliest proteins required for metanephric development, appears normal in both control and triple-mutant mice. Within the next 24 hours of development, the ureter normally invades the metanephric blastema, which subsequently condenses and becomes induced. In the triple-mutants, however, ureteric budding from the Wolffian duct fails to initiate and the metanephric blastema undergoes apoptosis by E11.5. This demonstrates that the Hox11 paralogs are critically important in the earliest processes of metanephric development. Further molecular characterization in this well-defined system should allow us to understand, more specifically, the role of these Hox11 paralogs in the development of this organ.