SummaryTo determine the fate of anti-DNA antibody-bearing B cells in normal mice, we generated transgenic mice bearing the heavy (H) and light (L) chain genes of a well-characterized anti-double-stranded DNA antibody. This antibody was originally isolated from a diseased MRL/Ipr mouse and has characteristics common to spontaneously arising anti-DNA antibodies. Results show that the H/L transgene (tg) immunoglobulin receptor is not expressed by animals bearing both tgs, although single tg animals (H or L) express their transgenes. Young H/L tg animals express few B cells, whereas adult H/L tg animals maintain almost normal B cell numbers. Analysis of the immunoglobulin receptors used by adult B cells shows that all contain the tg H chain in association with endogenous L chains. These B cells transcribe the L tg as well as the rearranged endogenous L chain gene, and loss of endogenous L chain gene transcription results in resurrection of the 3H9 H/L tg product. Examination of the endogenous L chains used by these cells shows that they represent a highly restricted subset of V genes. Taken together, these data suggest that autoreactive transgenic B cells can rearrange endogenous L chain genes to alter surface receptors. Those L chains that compete successfully with the L tg for H chain binding, and that create a nonautoreactive receptor, allow the B cell to escape deletion. We suggest that this receptor editing is a mechanism used by immature autoreactive B cells to escape tolerance. B cells expressing autoreactive Ig receptors are thought to be negatively regulated through inactivation or deletion. Inactivation was observed by Pike and Nossal (reviewed in reference 1), who demonstrated that low doses of antigen could render B cells specifically unresponsive to antigen and mitogen stimulation. This type of tolerance was shown in amplified form using mice with transgenes (tgs) 1 coding for an anti-hen egg lysozyme (HEL) antibody (reviewed in reference 2). These mice, when crossed with mice with a tg coding for HEL, yielded double-tg progeny whose B cells were refractory to antigenic or mitogenic stimulation. Deletion was dramatically demonstrated using a tg system in which genes of an anti-H-2 k antibody were introduced into mice of the H-2 k haplotype (reviewed in reference 3). These mice showed a great reduction in B cell number and absence of the tg idiotype on the remaining peripheral B cells.These types of negative regulation operate at the cellular level through uncoupling signaling pathways or by initiating cell death. Another level at which regulation might occur 1Abbreviations used in this paper: ANA, anti-nuclear antigen; ds, double stranded; tg, transgene.is by changing the specificity of antiself receptors (4). Such "editing" might occur at the genetic level by replacing V genes coding for antiself receptors with V genes coding for "harmless" receptors. Mechanisms are available at both the H and L chain g loci for V gene replacement. Here we describe another way of editing receptors. In this case, the L ...

The 5' flanking region of the mouse alpha-fetoprotein (AFP) gene contains a tissue-specific promoter and three upstream regulatory elements that behave as classical enhancers. At least one of these enhancers is now shown to be required for the tissue-specific expression of the AFP gene when it is introduced into the mouse genome by microinjection of cloned DNA fragments into fertilized eggs. Each enhancer can direct expression in the appropriate tissues, the visceral endoderm of the yolk sac, the fetal liver, and the gastrointestinal tract, but each exerts different influence in these three tissues. These differences may explain the tissue-specific diversity in the levels of expression characteristic of the AFP gene. The postnatal repression of transcription of the AFP gene in both liver and gut, as well as the reinitiation of its transcription during liver regeneration, is mimicked by the introduced gene when it is linked to the enhancer domains together or singly. Thus, the DNA sequence elements responsible for directing the activation of AFP transcription, its repression, and reinduction are contained in a limited segment of DNA within or 5' to the gene (or both) and are operative in the absence of the closely linked albumin gene.

The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. We expect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity. We discuss approaches for future research in the genetics of CPHD.

Mice homozygous for the recessive mutation vestigial tail (v-t), which arose spontaneously on Chromosome 11, exhibit vertebral abnormalities, including loss of caudal vertebrae leading to shortening of the tail. Wnt-3a, a member of the wingless family of secreted glycoproteins, maps to the same chromosome. Embryos homozygous for a null mutation in Wnt-3a (Wnt-3a "e°) have a complete absence of tail bud development and are truncated rostral to the hindlimbs. Several lines of evidence reveal that vt is a hypomorphic allele of Wnt-3a. We show that Wnt-3a and vt eosegregate in a high-resolution backcross and fail to complement, suggesting that Wnt-3a "~° and vt are allelic. Embryos heterozygous for both alleles have a phenotype intermediate between that of Wnt-3a n~° and vt homozygotes, lacking a tail, but developing thoracic and a variable number of lumbar vertebrae. Although no gross alteration in the Wnt-3a gene was detected in vt mice and the Wnt-3a coding region was normal, Wnt-3a expression was markedly reduced in vt/rt embryos consistent with a regulatory mutation in Wnt-3a. Furthermore, the analysis of allelic combinations indicates that Wnt-3a is required throughout the period of tail bud development for caudal somitogenesis. Interestingly, increasing levels of Wnt-3a activity appear to be necessary for the formation of more posterior derivatives of the paraxial mesoderm.

Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3′ transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.

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 coexpressed 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.

Recessive mutations in myosin 15, a class XV unconventional myosin, cause profound congenital deafness in humans and both deafness and vestibular dysfunction in mice homozygous for the shaker 2 and shaker 2(J) alleles. The shaker 2 allele is a previously described missense mutation of a highly conserved residue in the motor domain of myosin XV. The shaker 2(J) lesion, in contrast, is a 14.7 kb deletion that removes the last six exons from the 3"-terminus of the Myo15 transcript. These exons encode a FERM (F, ezrin, radixin and moesin) domain that may interact with integral membrane proteins. Despite the deletion of six exons, Myo15 mRNA transcripts and protein are present in the post-natal day 1 shaker 2(J) inner ear, which suggests that the FERM domain is critical for the development of normal hearing and balance. Myo15 transcripts are first detectable at embryonic day 13.5 in wild-type mice. Myo15 transcripts in the mouse inner ear are restricted to the sensory epithelium of the developing cristae ampularis, macula utriculi and macula sacculi of the vestibular system as well as to the developing organ of Corti. Both the shaker 2 and shaker 2(J) alleles result in abnormally short hair cell stereocilia in the cochlear and vestibular systems. This suggests that Myo15 may be important for both the structure and function of these sensory epithelia.

Pituitary thyrotropin (TSH) and gonadotropins (LH and FSH) are thought to be critical for thyroid and gonadal development and function. Each of these pituitary hormones is a heterodimer composed of a common a-subunit and unique ~-subunit, and heterodimerization is required for function. No mutations in the a-subunit or any of the 13-subunit genes have been reported in mice. To assess directly the functional role of TSH, LH, and FSH in thyroid and gonadal development, we created a disruption of the a-subunit gene by homologous recombination. The homozygous mutant animals were hypogonadal and exhibited profound hypothyroidism resulting in dwarfism. Thyroid development was arrested in late gestation, but GnRH neuron migration, development of secondary sex organs, and fetal and neonatal gonadal development were normal. This establishes the importance of thyrotropin in ontogeny and reveals that fetal pituitary gonadotropins are not required for sexual differentiation or genital development in male or female fetuses. The pituitary cells that produce TSH 13-subunit exhibited dramatic hypertrophy and hyperplasia as a result of the lack of thyroid function. This proliferative response occurred at the expense of somatotrope and lactotrope cells, consistent with a derivation of these three cell types from a common precursor.