Mark Solloway

A mouse knockout library for secreted and transmembrane proteins

Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

The Mammalian Cos2 Homolog Kif7 Plays an Essential Role in Modulating Hh Signal Transduction during Development

The Hedgehog (Hh) signaling pathway regulates development in animals ranging from flies to humans. Although its framework is conserved, differences in pathway components have been reported. A kinesin-like protein, Costal2 (Cos2), plays a central role in the Hh pathway in flies. Knockdown of a zebrafish homolog of Cos2, Kif7, results in ectopic Hh signaling, suggesting that Kif7 acts primarily as a negative regulator of Hh signal transduction. However, in vitro analysis of the function of mammalian Kif7 and the closely related Kif27 has led to the conclusion that neither protein has a role in Hh signaling. Using Kif7 knockout mice, we demonstrate that mouse Kif7, like its zebrafish and Drosophila homologs, plays a role in transducing the Hh signal. We show that Kif7 accumulates at the distal tip of the primary cilia in a Hh-dependent manner. We also demonstrate a requirement for Kif7 in the efficient localization of Gli3 to cilia in response to Hh and for the processing of Gli3 to its repressor form. These results suggest a role for Kif7 in coordinating Hh signal transduction at the tip of cilia and preventing Gli3 cleavage into a repressor form in the presence of Hh.

Wnt Isoform-Specific Interactions with Coreceptor Specify Inhibition or Potentiation of Signaling by LRP6 Antibodies

β-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states.

Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse

Mia3’s contribution to protein secretion is broader than previously realized—its absence impairs collagen deposition and normal development of cartilage and bone.

The Rfx4 Transcription Factor Modulates Shh Signaling by Regional Control of Ciliogenesis

Rfx4 regulates the formation of primary cilia, thereby playing a crucial role in central nervous system development. Regional Regulation of Cilia Formation Development of the central nervous system (CNS) requires the activation of transcriptional networks in a precise spatial and temporal pattern. Sonic hedgehog (Shh) signaling, which regulates the activity of the Gli family of transcriptional activators and repressors, is important for CNS development. Shh signaling involves a cellular structure called the primary cilium. Ashique et al. show that the transcription factor Rfx4 serves as a regionally specific regulator of the formation of the primary cilia in the developing CNS. Loss of its activity in mice results in aberrant Shh signaling and Gli3 activity, resulting in defective spinal cord and telencephalon development. Furthermore, the activity of Rfx4 may be regulated by phosphorylation, thus allowing Rfx4 to serve as an upstream regulator of Shh signaling and Gli3 activity in response to developmental signals. Regulatory factor X (Rfx) homologs regulate the transcription of genes necessary for ciliogenesis in invertebrates and vertebrates. Primary cilia are necessary for Hedgehog signaling and regulation of the activity of the transcriptional regulators known as Gli proteins, which are targets of Hedgehog signaling. Here, we describe an Rfx4L298P mouse mutant with distinct dorsoventral patterning defects in the ventral spinal cord and telencephalon due to aberrant Sonic hedgehog (Shh) signaling and Gli3 activity. We find that Ift172, which encodes an intraflagellar transport protein necessary for ciliogenesis, is a direct transcriptional target of Rfx4, and the decrease in its expression in the developing telencephalon and spinal cord of Rfx4L298P mutants correlates with defects in patterning and cilia formation. Our data indicate that Rfx4 is a regionally specific transcriptional regulator of ciliogenesis and thus is also a regionally specific modulator of Shh signaling during development of the central nervous system.

Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass

Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and β-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into β-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.

Chondroitin sulfate synthase 1 (Chsy1) is required for bone development and digit patterning.

Joint and skeletal development is highly regulated by extracellular matrix (ECM) proteoglycans, of which chondroitin sulfate proteoglycans (CSPGs) are a major class. Despite the requirement of joint CSPGs for skeletal flexibility and structure, relatively little is understood regarding their role in establishing joint positioning or in modulating signaling and cell behavior during joint formation. Chondroitin sulfate synthase 1 (Chsy1) is one of a family of enzymes that catalyze the extension of chondroitin and dermatan sulfate glycosaminoglycans. Recently, human syndromic brachydactylies have been described to have loss-of-function mutations at the CHSY1 locus. In concordance with these observations, we demonstrate that mice lacking Chsy1, though viable, display chondrodysplasia and decreased bone density. Notably, Chsy1(-/-) mice show a profound limb patterning defect in which orthogonally shifted ectopic joints form in the distal digits. Associated with the digit-patterning defect is a shift in cell orientation and an imbalance in chondroitin sulfation. Our results place Chsy1 as an essential regulator of joint patterning and provide a mouse model of human brachydactylies caused by mutations in CHSY1.

Wnt Signaling Is Regulated by Endoplasmic Reticulum Retention

Precise regulation of Wnt signaling is important in many contexts, as in development of the vertebrate forebrain, where excessive or ectopic Wnt signaling leads to severe brain defects. Mutation of the widely expressed oto gene causes loss of the anterior forebrain during mouse embryogenesis. Here we report that oto is the mouse ortholog of the gpi deacylase gene pgap1, and that the endoplasmic reticulum (ER)-resident Oto protein has a novel and deacylase-independent function during Wnt maturation. Oto increases the hydrophobicities of Wnt3a and Wnt1 by promoting the addition of glycophosphatidylinositol (gpi)-like anchors to these Wnts, which results in their retention in the ER. We also report that oto-deficient embryos exhibit prematurely robust Wnt activity in the Wnt1 domain of the early neural plate. We examine the effect of low oto expression on Wnt1 in vitro by knocking down endogenous oto expression in 293 and M14 melanoma cells using shRNA. Knockdown of oto results in increased Wnt1 secretion which is correlated with greatly enhanced canonical Wnt activity. These data indicate that oto deficiency increases Wnt signaling in vivo and in vitro. Finally, we address the mechanism of Oto-mediated Wnt retention under oto-abundant conditions, by cotransfecting Wnt1 with gpi-specific phospholipase D (GPI-PLD). The presence of GPI-PLD in the secretory pathway results in increased secretion of soluble Wnt1, suggesting that the gpi-like anchor lipids on Wnt1 mediate its retention in the ER. These data now provide a mechanistic framework for understanding the forebrain defects in oto mice, and support a role for Oto-mediated Wnt regulation during early brain development. Our work highlights a critical role for ER retention in regulating Wnt signaling in the mouse embryo, and gives insight into the notoriously inefficient secretion of Wnts.

Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these ‘non-homeostatic’ conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the ‘emergency circuit’ that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.