Retinoic Acid (RA) is a small lipophilic signaling molecule essential for embryonic development and adult tissue maintenance. Both an excess of RA and a deficiency of RA can cause pathogenic anomalies, hence it is critical to understand the mechanisms controlling the spatial and temporal distribution of RA. However, our current understanding of these processes remains incomplete. Vitamin A is metabolized to RA via two sequential enzymatic reactions. The first requires retinol dehydrogenase (RDH) activity to oxidize Vitamin A (retinol) to retinal, and the second requires retinaldehyde activity (RALDH) to oxidize retinal into RA. The first reaction has previously been attributed to the alcohol dehydrogenase (ADH) family, whose genes are ubiquitously or redundantly expressed. Consequently, the specificity of RA synthesis was thought to reside exclusively at the level of the second reaction. To better understand the metabolism of Vitamin A into RA during embryogenesis, we generated new mouse models that disrupt this process. Here we describe a new targeted knockout of Rdh10 in which RA synthesis is severely impaired, particularly at critical early embryonic stages. We also introduce a new mutant allele of Aldh1a2. Both mutations produce similar developmental defects resulting in lethality around embryonic day 10.5 (E10.5). The severity of the Rdh10 null phenotype demonstrates that embryonic oxidation of retinol is carried out primarily by RDH10 and that neither ADHs nor other enzymes contribute significantly to this reaction. We also show that reduced RA production results in upregulation of Rdh10. These data demonstrate that RDH10 plays a critical role in mediating the rate limiting RDH step of Vitamin A metabolism and functions as a nodal point in feedback regulation of RA synthesis. Moreover, RDH10-mediated oxidation of retinol plays as important a role in the control and regulation of RA production during embryogenesis as does the subsequent RALDH-mediated reaction.

Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843 Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and 35 S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843 Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies.

Cleavage of amyloid precursor protein (APP) by the Alzheimer's β-secretase (BACE1) is a key step in generating amyloid β-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with β-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by α-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.

Neural crest (NC) cell migration is crucial to the formation of peripheral tissues during vertebrate development. However, how NC cells respond to different microenvironments to maintain persistence of direction and cohesion in multicellular streams remains unclear. To address this, we profiled eight subregions of a typical cranial NC cell migratory stream. Hierarchical clustering showed significant differences in the expression profiles of the lead three subregions compared with newly emerged cells. Multiplexed imaging of mRNA expression using fluorescent hybridization chain reaction (HCR) quantitatively confirmed the expression profiles of lead cells. Computational modeling predicted that a small fraction of lead cells that detect directional information is optimal for successful stream migration. Single-cell profiling then revealed a unique molecular signature that is consistent and stable over time in a subset of lead cells within the most advanced portion of the migratory front, which we term trailblazers. Model simulations that forced a lead cell behavior in the trailing subpopulation predicted cell bunching near the migratory domain entrance. Misexpression of the trailblazer molecular signature by perturbation of two upstream transcription factors agreed with the in silico prediction and showed alterations to NC cell migration distance and stream shape. These data are the first to characterize the molecular diversity within an NC cell migratory stream and offer insights into how molecular patterns are transduced into cell behaviors.