Faculty Bibliography

BACKGROUND: The high affinity tyrosine kinase receptor, TrkB, is the primary receptor for brain derived neurotrophic factor (BDNF) and plays an important role in development, maintenance and plasticity of the striatal output medium size spiny neuron. The striatal BDNF/TrkB system is thereby implicated in many physiologic and pathophysiologic processes, the latter including mood disorders, addiction, and Huntington's disease. We crossed a mouse harboring a transgene directing cre-recombinase expression primarily to postnatal, dorsal striatal medium spiny neurons, to a mouse containing a floxed TrkB allele (fB) mouse designed for deletion of TrkB to determine its role in the adult striatum. RESULTS: We found that there were sexually dimorphic alterations in behaviors in response to stressful situations and drugs of abuse. Significant sex and/or genotype differences were found in the forced swim test of depression-like behaviors, anxiety-like behaviors on the elevated plus maze, and cocaine conditioned reward. Microarray analysis of dorsal striatum revealed significant dysregulation in individual and groups of genes that may contribute to the observed behavioral responses and in some cases, represent previously unidentified downstream targets of TrkB. CONCLUSIONS: The data point to a set of behaviors and changes in gene expression following postnatal deletion of TrkB in the dorsal striatum distinct from those in other brain regions.

BACKGROUND: A multitude of different approaches have been proposed for achieving optimal aesthetic results after nipple reconstruction. In contrast, however, only a few studies focus on the morbidity associated with this procedure, particularly after implant-based breast reconstruction. METHODS: Using a cross-sectional study design all patients who underwent implant-based breast reconstruction with subsequent nipple reconstruction between 2000 and 2010 at Stanford University Medical Center were identified. The aim of the study was to analyze the impact of the following parameters on the occurrence of postoperative complications: age, final implant volume, time interval from placement of final implant to nipple reconstruction, and history of radiotherapy. RESULTS: A total of 139 patients with a mean age of 47.5 years (range, 29 to 75 years) underwent 189 nipple reconstructions. The overall complication rate was 13.2 percent (N = 25 nipple reconstructions). No association was observed between age (p = 0.43) or implant volume (p = 0.47) and the occurrence of complications. A trend towards higher complication rates in patients in whom the time interval between final implant placement and nipple reconstruction was greater than 8.5 months was seen (p = 0.07). Radiotherapy was the only parameter that was associated with a statistically significant increase in postoperative complication rate (51.7 percent vs. 6.25 percent; p < 0.00001). CONCLUSION: While nipple reconstruction is a safe procedure after implant-based breast reconstruction in patients without a history of radiotherapy, the presence of an irradiated field converts it to a high-risk one with a significant increase in postoperative complication rate. Patients with a history of radiotherapy should be informed about their risk profile and as a result may choose autologous reconstruction instead. LEVEL OF EVIDENCE: IV.

Sharply delineated domains of cell types arise in developing tissues under instruction of inductive signal (morphogen) gradients, which specify distinct cell fates at different signal levels. The translation of a morphogen gradient into discrete spatial domains relies on precise signal responses at stable cell positions. However, cells in developing tissues undergoing morphogenesis and proliferation often experience complex movements, which may affect their morphogen exposure, specification, and positioning. How is a clear pattern achieved with cells moving around? Using in toto imaging of the zebrafish neural tube, we analyzed specification patterns and movement trajectories of neural progenitors. We found that specified progenitors of different fates are spatially mixed following heterogeneous Sonic Hedgehog signaling responses. Cell sorting then rearranges them into sharply bordered domains. Ectopically induced motor neuron progenitors also robustly sort to correct locations. Our results reveal that cell sorting acts to correct imprecision of spatial patterning by noisy inductive signals.

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1's predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.

The graded distribution of morphogens underlies many of the tissue patterns that form during development. How morphogens disperse from a localized source and how gradients in the target tissue form has been under debate for decades. Recent imaging studies and biophysical measurements have provided evidence for various morphogen transport models ranging from passive mechanisms, such as free or hindered extracellular diffusion, to cell-based dispersal by transcytosis or cytonemes. Here, we analyze these transport models using the morphogens Nodal, fibroblast growth factor and Decapentaplegic as case studies. We propose that most of the available data support the idea that morphogen gradients form by diffusion that is hindered by tortuosity and binding to extracellular molecules.

Large-scale genomics and computational approaches have identified thousands of putative long non-coding RNAs (lncRNAs). It has been controversial, however, as to what fraction of these RNAs is truly non-coding. Here, we combine ribosome profiling with a machine-learning approach to validate lncRNAs during zebrafish development in a high throughput manner. We find that dozens of proposed lncRNAs are protein-coding contaminants and that many lncRNAs have ribosome profiles that resemble the 5' leaders of coding RNAs. Analysis of ribosome profiling data from embryonic stem cells reveals similar properties for mammalian lncRNAs. These results clarify the annotation of developmental lncRNAs and suggest a potential role for translation in lncRNA regulation. In addition, our computational pipeline and ribosome profiling data provide a powerful resource for the identification of translated open reading frames during zebrafish development.

Animals are often in discrete behavioral states, but it is unclear how one specific state is generated and opposes alternative states. Flavell et al. now identify molecular and neural components in C. elegans that are involved in the generation of dwelling and roaming states.

Small molecules have been used since antiquity to regulate our sleep. Despite the explosion of diverse drugs to treat problems of too much or too little sleep, the detailed mechanisms of action and especially the neuronal targets by which these compounds alter human behavioural states are not well understood. Research efforts in model systems such as mouse, zebrafish and fruit fly are combining conditional genetics and optogenetics with pharmacology to map the effects of sleep-promoting drugs onto neural circuits. Recent studies raise the possibility that many small molecules alter sleep and wake via specific sets of critical neurons rather than through the global modulation of multiple brain targets. These findings also uncover novel brain areas as sleep/wake regulators and indicate that the development of circuit-selective drugs might alleviate sleep disorders with fewer side effects.

G protein-coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering of native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized mGluRs (LimGluRs). The light-agonized LimGluR2, on which we focused, was fast, bistable and supported multiple rounds of on/off switching. Light gated two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. We found that the light-antagonized tool LimGluR2-block was able to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalized the optical control to two additional family members: mGluR3 and mGluR6. This system worked in rodent brain slices and in zebrafish in vivo, where we found that mGluR2 modulated the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease.