The worldwide epidemic of obesity has increased the urgency of developing a deeper understanding of physiological systems related to energy balance and energy storage, including the mechanisms controlling the development of fat cells (adipocytes). The differentiation of committed preadipocytes to adipocytes is controlled by PPARγ and several other transcription factors 1, but the molecular basis for preadipocyte determination is not understood. Using a novel method for the quantitative analysis of transcriptional components, we identified the zinc-finger protein Zfp423 as a factor enriched in preadipose versus non-preadipose fibroblasts. Ectopic expression of Zfp423 in non-adipogenic NIH 3T3 fibroblasts robustly activates expression of PPARγ in undifferentiated cells and permits cells to undergo adipocyte differentiation under permissive conditions. ShRNA-mediated reduction of Zfp423 expression in 3T3-L1 cells blunts preadipocyte PPARγ expression and diminishes the ability of these cells to differentiate. Furthermore, both brown and white adipocyte differentiation is strikingly impaired in Zfp423-deficient mouse embryos. Zfp423 regulates PPARγ expression, in part, through amplification of the BMP signaling pathway, an effect dependent on the SMAD binding capacity of Zfp423. This study identifies Zfp423 as a transcriptional regulator of preadipocyte determination.

Defects in cilia are associated with several human disorders, including Kartagener syndrome 1 , polycystic kidney disease 2,3 , nephronophthisis 4 and hydrocephalus 5 . We proposed that the pleiotropic phenotype of Bardet-Biedl syndrome (BBS), which encompasses retinal degeneration, truncal obesity, renal and limb malformations and developmental delay, is due to dysfunction of basal bodies and cilia 6,7 . Here we show that individuals with BBS have partial or complete anosmia. To test whether this phenotype is caused by ciliary defects of olfactory sensory neurons, we examined mice with deletions of Bbs1 or Bbs4. Loss of function of either BBS protein affected the olfactory, but not the respiratory, epithelium, causing severe reduction of the ciliated border, disorganization of the dendritic microtubule network and trapping of olfactory ciliary proteins in dendrites and cell bodies. Our data indicate that BBS proteins have a role in the microtubule organization of mammalian ciliated cells and that anosmia might be a useful determinant of other pleiotropic disorders with a suspected ciliary involvement.BBS is caused by mutations in at least eight loci, seven of which have been identified 6,[8][9][10][11][12][13][14] . Although the sequences of the BBS proteins have not provided any clues to their function, BBS4, BBS5 and BBS8 are localized to the basal body of cultured cells and at ciliated borders in tissues 6,7,10 . In addition, all known orthologs of the mammalian BBS proteins are expressed specifically in ciliated sensory neurons in Caenorhabditis elegans 6,10 , raising the possibility that disruption of these proteins will lead to ciliary defects.Although the capacity to generate cilia is shared by most mammalian cells, some cells develop specialized cilia that mediate sensory function. The olfactory receptor neuron is a highly specialized example of a ciliated cell in which the apical process terminates in a complex structure, the dendritic knob, containing multiple basal bodies 15 . Eight or more immotile cilia emanate from this dendritic knob and extend more than 60 mm into the mucus. Given that at least three BBS proteins localize to the olfactory epithelium 6,7 , we considered that if ciliary defects underlie BBS, then olfactory structure and sensory function should be compromised in individuals with BBS. To test this hypothesis, we evaluated 19 individuals with BBS from 14 unrelated families using the fully validated, 12-item smell identification test. To compensate for varying degrees of visual impairment in subjects, each test was administered in a controlled setting by the same personnel. The test has a maximum possible score of 12. We compared the score of each individual with sex-derived normative data, ranked the relative degree of olfactory function by percentile and categorized olfactory function as normal (score of 9-12), abnormal (score of 8) or Ten individuals, including all three o15 y of age, scored in the normal range (9-12). Two subjects were abnormal (score of 8), and seven scored in th...

No abstract

SignificanceGenome editing, the introduction of precise changes in the genome, is revolutionizing our ability to decode the genome. Here we describe a simple method for genome editing in mammalian cells that takes advantage of an efficient mechanism for gene conversion that utilizes linear donors. We demonstrate that PCR fragments containing edits up to 1 kb require only 35-bp homology sequences to initiate repair of Cas9-induced double-stranded breaks in human cells and mouse embryos. We experimentally determine donor DNA design rules that maximize the recovery of edits without cloning or selection.

Bardet-Biedl syndrome (BBS) is a pleiotropic, heterogeneous human disease whose etiology lies primarily in dysfunctional basal bodies and/or cilia. Both BBS patients and several BBS mouse models exhibit impaired olfactory function. To explore the nature of olfactory defects in BBS, a genetic ablation of the mouse Bbs8 gene that incorporates a fluorescent reporter protein was created. The endogenous BBS8 protein and reporter are particularly abundant in olfactory sensory neurons (OSNs), and specific BBS8 antibodies reveal staining in the dendritic knob in a shell-like structure that surrounds the basal bodies. Bbs8-null mice have reduced olfactory responses to a number of odorants, and immunohistochemical analyses reveal a near-complete loss of cilia from OSNs and mislocalization of proteins normally enriched in cilia. To visualize altered protein localization in OSNs, we generated a SLP3 eGFP knock-in mouse and imaged the apical epithelium, including dendritic knobs and proximal cilia, in ex vivo tissue preparations. Additionally, protein reagents that reflect the characteristic neuronal activity of each OSN revealed altered activity in Bbs8-null cells. In addition to previously known defects at the ciliary border, we also observed aberrant targeting of OSN axons to the olfactory bulb; axons expressing the same receptor display reduced fasciculation and project to multiple targets in the olfactory bulb. We suggest that loss of BBS8 leads to a dramatic and variable reduction in cilia, the essential signaling platform for olfaction, which alters the uniformity of responses in populations of OSNs expressing the same receptor, thereby contributing to the observed axon-targeting defects.ciliopathy | olfactory activity | protein trafficking B ardet-Biedl syndrome (BBS), a heterogeneous human disease, encompasses pleiotropic phenotypes including obesity, polydactyly, retinal degeneration, and renal anomalies. The disease, associated with mutations in at least 16 genes, shows complex inheritance. The BBS8 gene was identified via shared homology with BBS4 and was recognized to bear similarity to bacterial pilF; pilF is thought to be involved in the assembly of pili, which are thin, hairlike extensions on prokaryotic cells (1). This prompted the hypothesis that BBS is primarily a disease of the basal body, a microtubule-based modified centriole that nucleates the ciliary axoneme. Subsequent work supports this common etiology of BBS (2). Characterized BBS genes are highly conserved exclusively among ciliated eukaryotes, and most BBS proteins localize to the basal body, centrosome, and/or cilium in ciliated cell-culture models and in ciliated tissues. BBS proteins are not thought to be essential structural proteins as the basal body and cilium remain largely intact in most mutant BBS models (3-5). Disruption of individual BBS genes leads to defects in intraflagellar transport (IFT), a process essential for protein trafficking within the cilium (6-8).Recent studies have found that seven BBS proteins-BBS1, -2, -4, -5, -7, -8,...

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only ~35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

The site of SARS-CoV-2 entry and replication critically impacts strategies for COVID-19 diagnosis, transmission mitigation, and treatment. We determined the cellular location of the SARS-CoV-2 target receptor protein, ACE2, in the human upper airway, finding striking enrichment (200-700 folds) in the olfactory neuroepithelium relative to nasal respiratory or tracheal epithelial cells. This cellular tropism of SARS-CoV-2 may underlie its high transmissibility and association with olfactory dysfunction, while suggesting a viral reservoir potentially amenable to intranasal therapy.

No abstract