PTH regulates fetal blood calcium and skeletal mineralization independently of PTHrP.

PTH and PTHrP both act in the regulation of fetal mineral metabolism. PTHrP regulates placental calcium transfer, fetal blood calcium, and differentiation of the cartilaginous growth plate into endochondral bone. PTH has been shown to influence fetal blood calcium, but its role in skeletal formation remains undefined. We compared skeletal morphology, mineralization characteristics, and gene expression in growth plates of fetal mice that lack parathyroids and PTH (Hoxa3 null) with the effects of loss of PTHrP (Pthrp null), loss of PTH/PTHrP receptor (Pthr1 null), and loss of both PTH and PTHrP (Hoxa3 null x Pthrp null). Loss of PTH alone does not affect morphology or gene expression in the skeletal growth plates, but skeletal mineralization and blood calcium are significantly reduced. In double-mutant fetuses (Hoxa3 null/Pthrp null), combined loss of PTH and PTHrP caused fetal growth restriction, limb shortening, greater reduction of fetal blood calcium, and reduced mineralization. These findings suggest that 1) PTH may play a more dominant role than PTHrP in regulating fetal blood calcium; 2) blood calcium and PTH levels are rate-limiting determinants of skeletal mineral accretion; and 3) lack of both PTH and PTHrP will cause fetal growth restriction.

Hoxb2 and hoxb4 act together to specify ventral body wall formation.

Three different alleles of the Hoxb4 locus were generated by gene targeting in mice. Two alleles contain insertions of a selectable marker in the first exon in either orientation, and, in the third, the selectable marker was removed, resulting in premature termination of the protein. Presence and orientation of the selectable marker correlated with the severity of the phenotype, indicating that the selectable marker induces cis effects on neighboring genes that influence the phenotype. Homozygous mutants of all alleles had cervical skeletal defects similar to those previously reported for Hoxb4 mutant mice. In the most severe allele, Hoxb4(PolII), homozygous mutants died either in utero at approximately E15.5 or immediately after birth, with a severe defect in ventral body wall formation. Analysis of embryos showed thinning of the primary ventral body wall in mutants relative to control animals at E11.5, before secondary body wall formation. Prior to this defect, both Alx3 and Alx4 were specifically down regulated in the most ventral part of the primary body wall in Hoxb4(PolII) mutants. Hoxb4(loxp) mutants in which the neo gene has been removed did not have body wall or sternum defects. In contrast, both the Hoxb4(PolII) and the previously described Hoxb2(PolII) alleles that have body wall defects have been shown to disrupt the expression of both Hoxb2 and Hoxb4 in cell types that contribute to body wall formation. Our results are consistent with a model in which defects in ventral body wall formation require the simultaneous loss of at least Hoxb2 and Hoxb4, and may involve Alx3 and Alx4.

Hoxa3 and pax1 regulate epithelial cell death and proliferation during thymus and parathyroid organogenesis.

The thymus and parathyroid glands in mice develop from a thymus/parathyroid primordium that forms from the endoderm of the third pharyngeal pouch. We investigated the molecular mechanisms that promote this unique process in which two distinct organs form from a single primordium, using mice mutant for Hoxa3 and Pax1. Thymic ectopia in Hoxa3(+/-)Pax1(-/-) compound mutants is due to delayed separation of the thymus/parathyroid primordium from the pharynx. The primordium is hypoplastic at its formation, and has increased levels of apoptosis. The developing third pouch in Hoxa3(+/-)Pax1(-/-) compound mutants initiates normal expression of the parathyroid-specific Gcm2 and thymus-specific Foxn1 genes. However, Gcm2 expression is reduced at E11.5 in Pax1(-/-) single mutants, and further reduced or absent in Hoxa3(+/-)Pax1(-/-) compound mutants. Subsequent to organ-specific differentiation from the shared primordium, both the parathyroids and thymus developed defects. Parathyroids in compound mutants were smaller at their formation, and absent at later stages. Parathyroids were also reduced in Pax1(-/-) mutants, revealing a new function for Pax1 in parathyroid organogenesis. Thymic hypoplasia at later fetal stages in compound mutants was associated with increased death and decreased proliferation of thymic epithelial cells. Our results suggest that a Hoxa3-Pax1 genetic pathway is required for both epithelial cell growth and differentiation throughout thymus and parathyroid organogenesis.

Gamma -secretase inhibitors repress thymocyte development.

A major therapeutic target in the search for a cure to the devastating Alzheimer's disease is gamma-secretase. This activity resides in a multiprotein enzyme complex responsible for the generation of Abeta42 peptides, precipitates of which are thought to cause the disease. Gamma-secretase is also a critical component of the Notch signal transduction pathway; Notch signals regulate development and differentiation of adult self-renewing cells. This has led to the hypothesis that therapeutic inhibition of gamma-secretase may interfere with Notch-related processes in adults, most alarmingly in hematopoiesis. Here, we show that application of gamma-secretase inhibitors to fetal thymus organ cultures interferes with T cell development in a manner consistent with loss or reduction of Notch1 function. Progression from an immature CD4-/CD8- state to an intermediate CD4+/CD8+ double-positive state was repressed. Furthermore, treatment beginning later at the double-positive stage specifically inhibited CD8+ single-positive maturation but did not affect CD4+ single-positive cells. These results demonstrate that pharmacological gamma-secretase inhibition recapitulates Notch1 loss in a vertebrate tissue and present a system in which rapid evaluation of gamma-secretase-targeted pharmaceuticals for their ability to inhibit Notch activity can be performed in a relevant context.

Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal pouch.

The thymus and parathyroids originate from a common primordium that develops from the third pharyngeal pouch in mice and humans. The molecular mechanism that specifies this primordium into distinct organ domains is not known. The Gcm2 and Foxn1 transcription factors are required for development of the parathyroid and thymus respectively, and are attractive candidates for this role. However, their embryonic expression patterns during pharyngeal pouch development and early thymus and parathyroid organogenesis have not been described. Here we report that Gcm2 is expressed specifically in the developing second and third pharyngeal pouches at E9.5, and is further confined to a small domain of the third pouch endoderm by E10.5. In contrast, Foxn1 is not expressed until after the common primordium is formed, beginning at E11.25. Our results show that Gcm2 and Foxn1 expression mark two complementary domains that prefigure parathyroid and thymus regions within the common primordium before morphological distinctions are present.

Fetal parathyroids are not required to maintain placental calcium transport.

We used Hoxa3 knockout mice and other mouse models to study the role of the fetal parathyroids in fetal calcium homeostasis. Hoxa3-null fetuses lack parathyroid glands, and absence of parathyroid hormone (PTH) was confirmed with a rodent PTH immunoradiometric assay. The ionized calcium level of Hoxa3-null fetuses was significantly lower than that of wild-type or heterozygous littermates or of the mother. Both the rate of placental calcium transfer and the plasma PTHrP level were normal in Hoxa3 mutants and their heterozygous siblings. Because we had previously observed an increase in placental calcium transfer in PTH/PTHrP receptor 1-null (Pthr1-null) fetuses, we assayed plasma PTHrP in those mice. Pthr1-null fetuses had plasma PTHrP levels 11-fold higher than those of their littermates. Northern analysis, immunohistochemical, and in situ hybridization studies of Pthr1-null fetuses indicated that liver and placenta had increased expression of PTHRP: In summary, loss of fetal parathyroids in Hoxa3-null fetuses caused marked hypocalcemia but did not alter placental calcium transfer or the circulating PTHrP level. The findings in the Pthr1-null fetuses indicate that several tissues may contribute to the circulating PTHrP level in fetal mice.

Thymus organogenesis and molecular mechanisms of thymic epithelial cell differentiation.

In the mature thymus, thymocyte maturation depends on interactions with different thymic epithelial subtypes in a three-dimensional thymic architecture. However, the molecular mechanisms that generate these epithelial subtypes are not well understood. Evidence is accumulating that during fetal thymus development, epithelial cells differentiate by successive interactions with differentiating thymocytes. This review presents fetal thymus development as a process of organogenesis, the main function of which is to promote thymic epithelial cell differentiation and the generation of a functional thymic microenvironment. In this model, endoderm-derived epithelial cells are the driving force in generating the thymic primordium, with hematopoietic cells providing later signals that organize and pattern the developing thymus.

Hoxa3 and pax1 transcription factors regulate the ability of fetal thymic epithelial cells to promote thymocyte development.

Thymocyte maturation into T cells depends on interactions between thymocytes and thymic epithelial cells. In this study, we show that mutations in two transcription factors, Hoxa3 and Pax1, act synergistically to cause defective thymic epithelial cell development, resulting in thymic ectopia and hypoplasia. Hoxa3+/-Pax1-/- compound mutant mice exhibited more severe thymus defects than Pax1-/- single mutants. Fetal liver adoptive transfer experiments revealed that the defect resided in radio-resistant stromal cells and not in hematopoietic cells. Compound mutants have fewer MHC class II+ epithelial cells, and the level of MHC expression detected was lower. Thymic epithelial cells in these mutants have reduced ability to promote thymocyte development, causing a specific block in thymocyte maturation at an early stage that resulted in a dramatic reduction in the number of CD4+8+ thymocytes. This phenotype was accompanied by increased apoptosis of CD4+8+ thymocytes and their immediate precursors, CD44-25-(CD3-4-8-) cells. Our results identify a transcriptional regulatory pathway required for thymic epithelial cell development and define multiple roles for epithelial cell regulation of thymocyte maturation at the CD4-8- to CD4+8+ transition.

Hox group 3 paralogs regulate the development and migration of the thymus, thyroid, and parathyroid glands.

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 +/-, Hoxb3-/-, Hoxd3-/-) 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.

Hox group 3 paralogous genes act synergistically in the formation of somitic and neural crest-derived structures.

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 with targeted disruption of Hoxb-1 fail to form the motor nucleus of the VIIth nerve.

Mice were generated with targeted disruptions in the hoxb-1 gene. Two separate mutations were created: the first disrupts only the homeodomain and the second inactivates the first exon as well as the homeodomain. The phenotypes associated with these two mutant alleles are indistinguishable in surviving adult mice. The predominant defect in these mutant mice is a failure to form the somatic motor component of the VIIth (facial) nerve, possibly through a failure to specify these neurons. The phenotype of hoxb-1 mutant homozygotes closely resembles features of the clinical profile associated with humans suffering from Bell's Palsy or Moebius Syndrome. These animals should therefore provide a useful animal model for these human diseases.

The role of Hoxa-3 in mouse thymus and thyroid development.

Targeted disruption of Hoxa-3 results in a number of regionally restricted defects in tissues and structures derived from or patterned by mesenchymal neural crest. However, analysis of mutant embryos with injections of a carbocyanine dye or with molecular markers that label these cells indicates that neither the amount nor the migration patterns of this neural crest population are grossly affected. Therefore, it appears that the loss of Hoxa-3 affects the intrinsic capacity of this neural crest cell population to differentiate and/or to induce proper differentiation of the surrounding pharyngeal arch and pouch tissues. Hoxa-3 mutant mice are athymic and show thyroid hypoplasia. Thymus development is first evident as an expansion of mesenchymal neural crest in the posterior part of the 3rd pharyngeal pouch. Prior to this expansion, a marked reduction in pax-1 expression is observed in these cells in the mutant embryos. As pax-1 mutant mice also show thymic hypoplasia, these results suggest that Hoxa-3 may be required to maintain pax-1 expression in these cells and that the reduction of pax-1 expression is part of the athymic teleology in Hoxa-3 mutant mice. The thyroid gland is formed from the fusion of two structures of separate embryonic origin, the thyroid diverticulum, which is formed from endodermal epithelium in the floor of the pharynx, and the ultimobranchial body, formed from mesenchymal neural crest in the 4th pharyngeal pouch. Both of these sites express Hoxa-3 and are defective in mutant mice. Often a vesicle is observed in mutant mice that is exclusively composed of calcitonin-producing cells, suggesting the persistence of an ultimobranchial body. Both aspects of the thyroid phenotype show variable expressivity among mutant animals, even on the two sides of the same mutant animal. This variability suggests the presence of a compensating gene or genes, whose utilization is stochastic. A reasonable candidate for providing this compensatory function is the paralogous gene Hoxb-3.

Loss of Hox-A1 (Hox-1.6) function results in the reorganization of the murine hindbrain.

Targeted disruption of the murine hox-A1 gene results in severe defects in the formation of the hindbrain and associated cranial ganglia and nerves. Carbocyanine dye injections were used to trace afferent and efferent projections to and from the hindbrain in hox-A1-/hox-A1- mutant mice. Defects were observed in the position of efferent neurons in the hindbrain and in their projection patterns. In situ hybridization was used to analyze the transcription pattern of genes expressed within specific rhombomeres. Krox-20, int-2 (fgf-3), and hox-B1 all display aberrant patterns of expression in hox-A1- mutant embryos. The observed morphological and molecular defects suggest that there are changes in the formation of the hindbrain extending from rhombomere 3 through rhombomere 8 including the absence of rhombomere 5. Also, motor neurons identified by their axon projection patterns which would normally be present in the missing rhombomere appear to be respecified to or migrate into adjacent rhombomeres, suggesting a role for hox-A1 in the specification of cell identity and/or cell migration in the hindbrain.

Two factors that bind to highly conserved sequences in mammalian type C retroviral enhancers.

The transcriptional enhancers of the Moloney and Friend murine leukemia viruses (MLV) are important determinants of viral pathogenicity. We used electrophoretic mobility shift and methylation interference assays to study nuclear factors which bind to a region of these enhancers whose sequence is identical between Moloney and Friend viruses and particularly highly conserved among 35 mammalian type C retroviruses whose enhancer sequences have been aligned (E. Golemis, N. A. Speck, and N. Hopkins, J. Virol. 64:534-542, 1990). Previous studies identified sites for the leukemia virus factor b (LVb) and core proteins in this region (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987) as well as a site, overlapping those for LVb and core, for a third factor (N. R. Manley, M. A. O'Connell, P. A. Sharp, and N. Hopkins, J. Virol. 63:4210-4223, 1989). Surprisingly, the latter factor appeared to also bind two sites identified in the Friend MLV enhancer, Friend virus factor a and b1 (FVa and FVb1) sites, although the sequence basis for the ability of the protein to bind these diverse sites was not apparent. Here we describe the further characterization of this binding activity, termed MCREF-1 (for mammalian type C retrovirus enhancer factor 1), and the identification of a consensus sequence for its binding, GGN8GG. We also identify a factor, abundant in mouse T-cell lines and designated LVt, which binds to two sites in the Moloney MLV enhancer, overlapping the previously identified LVb and LVc binding sites. These sites contain the consensus binding site for the Ets family of proteins. We speculate on how distinct arrays of these factors may influence the disease-inducing phenotype.

Nuclear factors that bind to the enhancer region of nondefective Friend murine leukemia virus.

Nondefective Friend murine leukemia virus (MuLV) causes erythroleukemia when injected into newborn NFS mice, while Moloney MuLV causes T-cell lymphoma. Exchange of the Friend virus enhancer region, a sequence of about 180 nucleotides including the direct repeat and a short 3'-adjacent segment, for the corresponding region in Moloney MuLV confers the ability to cause erythroid disease on Moloney MuLV. We have used the electrophoretic mobility shift assay and methylation interference analysis to identify cellular factors which bind to the Friend virus enhancer region and compared these with factors, previously identified, that bind to the Moloney virus direct repeat (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987). We identified five binding sites for sequence-specific DNA-binding proteins in the Friend virus enhancer region. While some binding sites are present in both the Moloney and Friend virus enhancers, both viruses contain unique sites not present in the other. Although none of the factors identified in this report which bind to these unique sites are present exclusively in T cells or erythroid cells, they bind to three regions of the enhancer shown by genetic analysis to encode disease specificity and thus are candidates to mediate the tissue-specific expression and distinct disease specificities encoded by these virus enhancer elements.