Sally A. Camper

The α(1.3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E- and P-selectin ligand biosynthesis.

alpha(1,3)Fucosylated oligosaccharides represent components of leukocyte counterreceptors for E- and P-selectins and of L-selectin ligands expressed by lymph node high endothelial venules (HEV). The identity of the alpha(1,3)fucosyltransferase(s) required for their expression has been uncertain, as has a requirement for alpha(1,3)fucosylation in HEV L-selectin ligand activity. We demonstrate here that mice deficient in alpha(1,3) fucosyltransferase Fuc-TVII exhibit a leukocyte adhesion deficiency characterized by absent leukocyte E- and P-selectin ligand activity and deficient HEV L-selectin ligand activity. Selectin ligand deficiency is distinguished by blood leukocytosis, impaired leukocyte extravasation in inflammation, and faulty lymphocyte homing. These observations demonstrate an essential role for Fuc-TVII in E-, P-, and L-selectin ligand biosynthesis and imply that this locus can control leukocyte trafficking in health and disease.

Role of Ahch in gonadal development and gametogenesis

Ahch (also known as Dax1) encodes a transcription factor that has been implicated in sex determination and gonadal differentiation. Mutations in human AHC cause X-linked, adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Duplication of the Xp21 dosage-sensitive sex reversal (DSS) region, which contains the Ahch locus, and transgenic overexpression of Ahch (ref. 6) cause male-to-female sex reversal. Using Cre-mediated disruption of Ahch, we have generated a mouse model of AHC-HH that allows the function of Ahch to be examined in both males and females. Although Ahch has been postulated to function as an ovarian determination gene, the loss of Ahch function in females does not affect ovarian development or fertility. Ahch is instead essential for the maintenance of spermatogenesis. Lack of Ahch causes progressive degeneration of the testicular germinal epithelium independent of abnormalities in gonadotropin and testosterone production and results in male sterility. Ahch is thus not an ovarian determining gene, but rather has a critical role in spermatogenesis.

SnoRNA Snord116 (Pwcr1/MBII-85) Deletion Causes Growth Deficiency and Hyperphagia in Mice

Prader-Willi syndrome (PWS) is the leading genetic cause of obesity. After initial severe hypotonia, PWS children become hyperphagic and morbidly obese, if intake is not restricted. Short stature with abnormal growth hormone secretion, hypogonadism, cognitive impairment, anxiety and behavior problems are other features. PWS is caused by lack of expression of imprinted genes in a ∼4 mb region of chromosome band 15q11.2. Our previous translocation studies predicted a major role for the C/D box small nucleolar RNA cluster SNORD116 (PWCR1/HBII-85) in PWS. To test this hypothesis, we created a ∼150 kb deletion of the >40 copies of Snord116 (Pwcr1/MBII-85) in C57BL/6 mice. Snord116del mice with paternally derived deletion lack expression of this snoRNA. They have early-onset postnatal growth deficiency, but normal fertility and lifespan. While pituitary structure and somatotrophs are normal, liver Igf1 mRNA is decreased. In cognitive and behavior tests, Snord116del mice are deficient in motor learning and have increased anxiety. Around three months of age, they develop hyperphagia, but stay lean on regular and high-fat diet. On reduced caloric intake, Snord116del mice maintain their weight better than wild-type littermates, excluding increased energy requirement as a cause of hyperphagia. Normal compensatory feeding after fasting, and ability to maintain body temperature in the cold indicate normal energy homeostasis regulation. Metabolic chamber studies reveal that Snord116del mice maintain energy homeostasis by altered fuel usage. Prolonged mealtime and increased circulating ghrelin indicate a defect in meal termination mechanism. Snord116del mice, the first snoRNA deletion animal model, reveal a novel role for a non-coding RNA in growth and feeding regulation.

The Pit-1 transcription factor gene is a candidate for the murine Snell dwarf mutation

Two nonallelic mouse mutations with severe dwarf phenotypes are characterized by a lack of growth hormone, prolactin, and thyroid stimulating hormone. The cells that normally synthesize these pituitary hormones express a common transcription factor called GHF-1 or Pit-1. Using an intersubspecific backcross, we have demonstrated tight linkage of the Pit-1 and Snell dwarf (dw) genes on mouse chromosome 16. No recombination was observed between Pit-1 and dw in 110 individuals examined. Southern blot analysis of genomic DNA reveals that the Pit-1 gene is rearranged in C3H/HeJ-dwJ/dw mice but not in coisogenic +/+ animals, providing molecular evidence that a lesion in the Pit-1 gene results in the Snell dwarf phenotype. Demonstration of low levels of Pit-1 expression in Ames dwarf (df) mice implies that both Pit-1 and df expression may be required for pituitary differentiation.

How many homeobox genes does it take to make a pituitary gland

The secrets of anterior pituitary development and cell lineage determination have been revealed mostly by genetic analyses. The requirement for three homeobox genes, Lbx3, Lbx4 and Titf1, during early organogenesis was proven by gene targeting. Spontaneous mouse mutations revealed two additional homeobox genes, Pit1 and Prop1, that are critical for specialization and proliferation of subsets of the five differentiated cell types. Analysis of patients with pituitary insufficiency has demonstrated the importance of these two genes in human pituitary function. Recently, several other homeobox genes have been identified and implicated in pituitary organogenesis. Genetic manipulations of these genes will undoubtedly add to the emerging genetic hierarchy regulating the ontogeny of this major hormone-producing gland.

The bicoid -related Pitx gene family in development

The important roles of homeobox genes in development of the hindbrain and axial body are well established. More recently, it has become clear that certain subfamilies of homeobox genes play particularly important roles in the development of more anterior structures. These have included the paired gene family in the eye (Gehring, 1996; Hanson and Van Heyningen, 1995; Macdonald and Wilson, 1996; Wehr and Gruss, 1996), the orthodenticle and distalless gene families in the foreand midbrains (Acampora et al., 1996; Acampora et al., 1995; Price et al., 1991; Simeone et al., 1994; Williams, 1998), and the Lhx gene family in the pituitary gland (Sheng et al., 1997; Sheng et al., 1996). This review summarizes the newly identified Pitx gene family and its role in development. This family includes three vertebrate paralogues that have been cloned in multiple organisms, and a fly cognate. Mutations in two members of this gene family lead to human disease or birth defects affecting anterior structures. The nomenclature for this gene family has been complicated by the fact that members have been cloned and uniquely named by more than one laboratory (Table 1). The first member of this family, mouse Ptx1 (pituitary homeobox 1) was isolated as a transcription factor involved in pro-opiomelanocortin gene transcription in anterior pituitary corticotropes (Lamonerie et al., 1996). However, since some pentaxin genes in mouse and human had previously been assigned the Ptx gene symbol, the gene symbols for the three mouse paralogues for this new homeobox gene family are Pitx1, Pitx2, and Pitx3 (Mouse Genome Database). In this review, we have adopted the official nomenclature of the MGD and propose that, for clarity, this nomenclature be adopted for other organisms. Three vertebrate paralogues, Pitx1, Pitx2, and Pitx3, have all been cloned from mouse and human (Table 1 and references therein). Some paralogues have also been cloned from chicken (Pitx1 and Pitx2), xenopus and zebrafish (Pitx2), and rat (Pitx3) (Table 1 and references therein). In two reports, mouse Pitx1 was cloned in functional assays: in a two-hybrid screen using Pit-1 as bait (Szeto et al., 1996) and as noted above. Human PITX2 was identified by positional cloning of the Rieger Syndrome gene (Semina et al., 1996). In the other reports, cloning was the result of using degenerate PCR or low stringency hybridization to detect expressed homeobox sequences in a variety of embryonic and adult tissues. The difficulty in cloning Pitx1 from xenopus and zebrafish has suggested that this orthologue may not be as widely distributed in nature as Pitx2 (Kitamura et al., 1997). However, the recent identification of a fly Pitx gene during a chromosome walk demonstrates that this gene family arose prior to the divergence of vertebrates and invertebrates (Vorbruggen et al., 1997). Each vertebrate paralogue has been mapped genetically in mouse and human (Table 1). The Pitx proteins all belong to the bicoid-related subclass of homeodomain proteins because they encode the defining lysine at residue 50 within the homeodomain. This residue, at residue 9 within the recognition helix of the homeodomain, is the major determinant of DNA binding specificity (Gehring et al., 1994; Hanes and Brent, 1989). Several members of this small subfamily are essential for axis and pattern formation (Ang et al., 1996). Pitx2 expresses multiple protein isoforms as a result of alternative splicing (Gage and Camper, 1997; Kitamura et al., 1997) and the use of different promoters (P. Gage and E. Semina, unpublished results) (Fig. 1). The three vertebrate paralogues are all highly conserved at the amino acid level (Fig. 1). For example, in mouse the Pitx2 and Pitx3 homeodomains are identical while Pitx1 differs by only two amino acids. The paralogues are also conserved Cterminal to the homeodomain (55–70%). In contrast, the N-termini of these proteins are essentially unrelated. The vertebrate orthologues are even more highly conserved. For example, there the mouse and chicken Pitx2a proteins are 96% identical with only ten amino acid substitutions between them. The Drosophila Pitx protein shows high conservation to the vertebrate proteins within the homeodomain (90–93%) and a short region near C-terminus that has been termed the OAR sequence (Furukawa et al., 1997) or the C-peptide (Kitamura et al., 1997). This sequence is present in several homeobox genes. In Pitx2, this domain appears to function as an intrinsic inhibitor of DNA binding activity whose function can be modulated by protein-protein interactions (Amendt et al., 1998). The vertebrate Pitx genes each have unique developmental and tissue-specific expression patterns (Fig. 2 and Table 2). However, there are several significant overlaps in expression pattern (Fig. 2). The most significant may be in the eye, where both Pitx2 and Pitx3 are expressed in the mesenchyme and its derivatives (Semina et al., 1998; Semina et al., 1996; Smidt et al., 1997). Demonstration in humans that mutations to Pitx2 result in Rieger’s Syndrome (Semina et al., 1996) and mutations to Pitx3 result in anterior segment mesenchymal dysgenesis and dominant cataracts (Semina et al., 1998) confirmed the importance of these genes in eye development. These autosomal-dominant conditions each affect the development or maintenance of anterior structures of the eye. Interestingly, mouse Pitx3 maps near aphakia, a recessive mutation resulting in small eyes that lack lenses and fail to develop beyond 11 days of gestation (Semina et al., 1997). Rieger’s Syndrome patients frequently show defects in dental and umbilical development in addition to their ocular defects (Feingold et al., 1969; Rieger, 1935), and subsets of patients also present with isolated growth insufficiency (Feingold et al., 1969). Several observations suggest that Pitx genes are also important for the development and function of other organs. The stomodeum is an ectoderm-derived layer of epithelium that derives from the anterior neural ridge and forms the earliest mouth structures (Couly and Le Douarin, 1985). Pitx1 expression defines the stomodeum and continues within stomodial derivatives, including the nasal pit and Rathke’s pouch (Lanctot et al., 1997). Pitx1 is also expressed more caudally in the posterior lateral plate and extraCorrespondence to: P.J. Gage Mammalian Genome 10, 197–200 (1999).

Pitx1 and Pitx2 are required for development of hindlimb buds

Two closely related homeobox transcription factors, Pitx1 and Pitx2, have been implicated in patterning of lateral plate mesoderm derivatives: Pitx1 for specification of hindlimb identity and Pitx2 for determination of laterality. We show that, together, Pitx1 and Pitx2 are required for formation of hindlimb buds and, when present in limited doses, for development of proximal (femur) and anterior (tibia and digit 1) hindlimb structures. Although Pitx1 is expressed throughout developing hindlimb buds, Pitx2 is not expressed in limb bud mesenchyme itself, but is co-expressed with Pitx1 in the presumptive hindlimb field before bud growth. Thus, Pitx1 and Pitx2 genes are required for sustained hindlimb bud growth and formation of hindlimbs.

Molecular mechanisms of pituitary organogenesis: In search of novel regulatory genes

Defects in pituitary gland organogenesis are sometimes associated with congenital anomalies that affect head development. Lesions in transcription factors and signaling pathways explain some of these developmental syndromes. Basic research studies, including the characterization of genetically engineered mice, provide a mechanistic framework for understanding how mutations create the clinical characteristics observed in patients. Defects in BMP, WNT, Notch, and FGF signaling pathways affect induction and growth of the pituitary primordium and other organ systems partly by altering the balance between signaling pathways. The PITX and LHX transcription factor families influence pituitary and head development and are clinically relevant. A few later-acting transcription factors have pituitary-specific effects, including PROP1, POU1F1 (PIT1), and TPIT (TBX19), while others, such as NeuroD1 and NR5A1 (SF1), are syndromic, influencing development of other endocrine organs. We conducted a survey of genes transcribed in developing mouse pituitary to find candidates for cases of pituitary hormone deficiency of unknown etiology. We identified numerous transcription factors that are members of gene families with roles in syndromic or non-syndromic pituitary hormone deficiency. This collection is a rich source for future basic and clinical studies.

Gata2 is required for HSC generation and survival

GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition and thereafter for HSC survival

Thyroid hormone resistance and increased metabolic rate in the RXR-γ-deficient mouse

Vitamin A and retinoids affect pituitary-thyroid function through suppression of serum thyroid-stimulating hormone (TSH) levels and TSH-β subunit gene expression. We have previously shown that retinoid X receptor–selective (RXR-selective) ligands can suppress serum TSH levels in vivo and TSH-β promoter activity in vitro. The RXR-γ isotype has limited tissue distribution that includes the thyrotrope cells of the anterior pituitary gland. In this study, we have performed a detailed analysis of the pituitary-thyroid function of mice lacking the gene for the RXR-γ isotype. These mice had significantly higher serum T4 levels and TSH levels than did wild-type (WT) controls. Treatment of RXR-γ–deficient and WT mice with T3 suppressed serum TSH and T4 levels in both groups, but RXR-γ–deficient mice were relatively resistant to exogenous T3. RXR-γ–deficient mice had significantly higher metabolic rates than did WT controls, suggesting that these animals have a pattern of central resistance to thyroid hormone. RXR-γ, which is also expressed in skeletal muscle and the hypothalamus, may have a direct effect on muscle metabolism, regulation of food intake, or thyrotropin-releasing hormone levels in the hypothalamus. In conclusion, the RXR-γ isotype appears to contribute to the regulation of serum TSH and T4 levels and to affect peripheral metabolism through regulation of the hypothalamic-pituitary-thyroid axis or through direct effects on skeletal muscle.