Catherine Guenther

Human-specific loss of regulatory DNA and the evolution of human-specific traits

Humans differ from other animals in many aspects of anatomy, physiology, and behaviour; however, the genotypic basis of most human-specific traits remains unknown. Recent whole-genome comparisons have made it possible to identify genes with elevated rates of amino acid change or divergent expression in humans, and non-coding sequences with accelerated base pair changes. Regulatory alterations may be particularly likely to produce phenotypic effects while preserving viability, and are known to underlie interesting evolutionary differences in other species. Here we identify molecular events particularly likely to produce significant regulatory changes in humans: complete deletion of sequences otherwise highly conserved between chimpanzees and other mammals. We confirm 510 such deletions in humans, which fall almost exclusively in non-coding regions and are enriched near genes involved in steroid hormone signalling and neural function. One deletion removes a sensory vibrissae and penile spine enhancer from the human androgen receptor (AR) gene, a molecular change correlated with anatomical loss of androgen-dependent sensory vibrissae and penile spines in the human lineage. Another deletion removes a forebrain subventricular zone enhancer near the tumour suppressor gene growth arrest and DNA-damage-inducible, gamma (GADD45G), a loss correlated with expansion of specific brain regions in humans. Deletions of tissue-specific enhancers may thus accompany both loss and gain traits in the human lineage, and provide specific examples of the kinds of regulatory alterations and inactivation events long proposed to have an important role in human evolutionary divergence.

A molecular basis for classic blond hair color in Europeans

Hair color differences are among the most obvious examples of phenotypic variation in humans. Although genome-wide association studies (GWAS) have implicated multiple loci in human pigment variation, the causative base-pair changes are still largely unknown. Here we dissect a regulatory region of the KITLG gene (encoding KIT ligand) that is significantly associated with common blond hair color in northern Europeans. Functional tests demonstrate that the region contains a regulatory enhancer that drives expression in developing hair follicles. This enhancer contains a common SNP (rs12821256) that alters a binding site for the lymphoid enhancer-binding factor 1 (LEF1) transcription factor, reducing LEF1 responsiveness and enhancer activity in cultured human keratinocytes. Mice carrying ancestral or derived variants of the human KITLG enhancer exhibit significant differences in hair pigmentation, confirming that altered regulation of an essential growth factor contributes to the classic blond hair phenotype found in northern Europeans.

Dual hindlimb control elements in the Tbx4 gene and region-specific control of bone size in vertebrate limbs.

The Tbx4 transcription factor is crucial for normal hindlimb and vascular development, yet little is known about how its highly conserved expression patterns are generated. We have used comparative genomics and functional scanning in transgenic mice to identify a dispersed group of enhancers controlling Tbx4 expression in different tissues. Two independent enhancers control hindlimb expression, one located upstream and one downstream of the Tbx4 coding exons. These two enhancers, hindlimb enhancer A and hindlimb enhancer B (HLEA and HLEB), differ in their primary sequence, in their precise patterns of activity within the hindlimb, and in their degree of sequence conservation across animals. HLEB is highly conserved from fish to mammals. Although Tbx4 expression and hindlimb development occur at different axial levels in fish and mammals, HLEB cloned from either fish or mouse is capable of driving expression at the appropriate position of hindlimb development in mouse embryos. HLEA is highly conserved only in mammals. Deletion of HLEA from the endogenous mouse locus reduces expression of Tbx4 in the hindlimb during embryogenesis, bypasses the embryonic lethality of Tbx4-null mutations, and produces viable, fertile mice with characteristic changes in the size of bones in the hindlimb but not the forelimb. We speculate that dual hindlimb enhancers provide a flexible genomic mechanism for altering the strength and location of Tbx4 expression during normal development, making it possible to separately modify the size of forelimb and hindlimb bones during vertebrate evolution.

Mineral formation in joints caused by complete or joint-specific loss of ANK function.

To reveal the ANK complete loss of function phenotype in mice, we generated conditional and null alleles. Mice homozygous for the null allele exhibited widespread joint mineralization, similar in severity to animals harboring the original ank allele. A delayed yet similar phenotype was observed in mice with joint‐specific loss of ANK function.

Evolving New Skeletal Traits by cis-Regulatory Changes in Bone Morphogenetic Proteins.

Changes in bone size and shape are defining features of many vertebrates. Here we use genetic crosses and comparative genomics to identify specific regulatory DNA alterations controlling skeletal evolution. Armor bone-size differences in sticklebacks map to a major effect locus overlapping BMP family member GDF6. Freshwater fish express more GDF6 due in part to a transposon insertion, and transgenic overexpression of GDF6 phenocopies evolutionary changes in armor-plate size. The human GDF6 locus also has undergone distinctive regulatory evolution, including complete loss of an enhancer that is otherwise highly conserved between chimps and other mammals. Functional tests show that the ancestral enhancer drives expression in hindlimbs but not forelimbs, in locations that have been specifically modified during the human transition to bipedalism. Both gain and loss of regulatory elements can localize BMP changes to specific anatomical locations, providing a flexible regulatory basis for evolving species-specific changes in skeletal form.

Shaping Skeletal Growth by Modular Regulatory Elements in the Bmp5 Gene

Cartilage and bone are formed into a remarkable range of shapes and sizes that underlie many anatomical adaptations to different lifestyles in vertebrates. Although the morphological blueprints for individual cartilage and bony structures must somehow be encoded in the genome, we currently know little about the detailed genomic mechanisms that direct precise growth patterns for particular bones. We have carried out large-scale enhancer surveys to identify the regulatory architecture controlling developmental expression of the mouse Bmp5 gene, which encodes a secreted signaling molecule required for normal morphology of specific skeletal features. Although Bmp5 is expressed in many skeletal precursors, different enhancers control expression in individual bones. Remarkably, we show here that different enhancers also exist for highly restricted spatial subdomains along the surface of individual skeletal structures, including ribs and nasal cartilages. Transgenic, null, and regulatory mutations confirm that these anatomy-specific sequences are sufficient to trigger local changes in skeletal morphology and are required for establishing normal growth rates on separate bone surfaces. Our findings suggest that individual bones are composite structures whose detailed growth patterns are built from many smaller lineage and gene expression domains. Individual enhancers in BMP genes provide a genomic mechanism for controlling precise growth domains in particular cartilages and bones, making it possible to separately regulate skeletal anatomy at highly specific locations in the body.

The progressive ankylosis protein regulates cementum apposition and extracellular matrix composition.

Background/Aims: Tooth root cementum is sensitive to modulation of inorganic pyrophosphate (PPi), an inhibitor of hydroxyapatite precipitation. Factors increasing PPi include progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) while tissue nonspecific alkaline phosphatase hydrolyzes PPi. Studies here aimed to define the role of ANK in root and cementum by analyzing tooth development in Ank knock-out (KO) mice versus wild type. Materials and Methods: Periodontal development in KO versus control mice was analyzed by histology, histomorphometry, immunohistochemistry, in situ hybridization, electron microscopy, and nanoindentation. Cementoblast cultures were used in vitro to provide mechanistic underpinnings for PPi modulation of cell function. Results: Over the course of root development, Ank KO cervical cementum became 8- to 12-fold thicker than control cervical cementum. Periodontal ligament width was maintained and other dentoalveolar tissues, including apical cementum, were unaltered. Cervical cementum uncharacteristically included numerous cells, from rapid cementogenesis. Ank KO increased osteopontin and dentin matrix protein 1 gene and protein expression, and markedly increased NPP1 protein expression in cementoblasts but not in other cell types. Conditional ablation of Ank in joints and periodontia confirmed a local role for ANK in cementogenesis. In vitro studies employing cementoblasts indicated that Ank and Enpp1 mRNA levels increased in step with mineral nodule formation, supporting a role for these factors in regulation of cementum matrix mineralization. Conclusion: ANK, by modulating local PPi, controls cervical cementum apposition and extracellular matrix. Loss of ANK created a local environment conducive to rapid cementogenesis; therefore, approaches modulating PPi in periodontal tissues have potential to promote cementum regeneration.

Over‐expression of BMP4 and BMP5 in a child with axial skeletal malformations and heterotopic ossification: A new syndrome

Bone morphogenetic proteins (BMPs) are a highly conserved class of signaling molecules that induce ectopic cartilage and bone formation in vivo. Dysregulated expression of bone morphogenetic protein 4 (BMP4) is found in the cells of patients who have fibrodysplasia ossificans progressiva (FOP), a genetic disorder of axial and appendicular skeletal malformation and progressive heterotopic ossification. Loss of function mutations in the bone morphogenetic protein 5 (bmp5) gene leading to under‐expression of BMP5 cause the murine short ear syndrome, characterized by small malformed ears and a broad range of axial skeletal malformations. We found features reminiscent of both the short ear mouse and FOP in a child with malformed external ears, multiple malformations of the axial skeleton, and progressive heterotopic ossification in the neck and back. We examined BMP mRNA expression in transformed lymphocytes by semi‐quantitative RT‐PCR and protein expression by ELISA assays and immunohistochemistry. Elevated levels of BMP4 and BMP5 mRNA and protein were detected in the patients cells while levels of BMP2 mRNA were unchanged. Our data suggest that dysregulated expression of BMP4 and BMP5 genes is associated with an array of human axial skeletal abnormalities similar to the short ear mouse and FOP.

The Phosphate Exporter xpr1b Is Required for Differentiation of Tissue-Resident Macrophages

Phosphate concentration is tightly regulated at the cellular and organismal levels. The first metazoan phosphate exporter, XPR1, was recently identified, but its in vivo function remains unknown. In a genetic screen, we identified a mutation in a zebrafish ortholog of human XPR1, xpr1b. xpr1b mutants lack microglia, the specialized macrophages that reside in the brain, and also displayed an osteopetrotic phenotype characteristic of defects in osteoclast function. Transgenic expression studies indicated that xpr1b acts autonomously in developing macrophages. xpr1b mutants display no gross developmental defects that may arise from phosphate imbalance. We constructed a targeted mutation of xpr1a, a duplicate of xpr1b in the zebrafish genome, to determine whether Xpr1a and Xpr1b have redundant functions. Single mutants for xpr1a were viable, and double mutants for xpr1b;xpr1a were similar to xpr1b single mutants. Our genetic analysis reveals a specific role for the phosphate exporter Xpr1 in the differentiation of tissue macrophages.