Deciphering ret signalling in cell biology and development

The rearranged during transformation (RET) tyrosine kinase regulates a plethora of biological processes such as cell survival, proliferation and migration and is essential for the normal development of several organs such as the sensory, enteric and sympathetic nervous systems and the kidneys. After RET activation by its ligands several intracellular tyrosine residues are phosphorylated and serve as binding sites for adaptor proteins that activate different downstream signalling pathways. One prominent binding site is tyrosine 1062. This residue is part of a binding motif for the phosphotyrosine binding (PTB) proteins DOK1-6, FRS2 and SHCA,B,C. The binding of PTB adaptors depends on the amino acids N-terminal of the tyrosine, and this feature can be utilised to engineer adaptor-specific receptors. RET is known to be recruited into cholesterol-rich membrane domains upon activation, but the mechanism and biological importance of this translocation were previously unknown. In Paper I, we analyse the influence of the membrane domain localization of RET and its adaptors on their signalling characteristics. We show that the lipid raft-associated FRS2 recruits RET to lipid raft domains, while SHC localizes it to other membrane regions. A lipid raft-bound SHC (SHCMLS) resembles FRS2 both in signalling, translocation of RET and biological functionality, with diminished support of cell survival and increased migration of SHCMLS compared to normal SHC. In contrast to SHC, both FRS2 and SHCMLS functions depend on lipid raft integrity. RET signalling is important for the development of several organ systems. In particular Y1062 plays a role in both the enteric and sympathetic nervous system and in nephrogenesis, however the specific roles of the different Y1062 binding proteins in vivo were unknown. In Paper II I investigate the role of RET signalling via DOK, FRS2 or SHC from Y1062 in vivo. Ret9Frs/9Frs mice show severe enteric aganglionosis, reduced soma size of dorsal root ganglion (DRG) neurons and mechanical hypersensitivity at early postnatal stages. Ret9Shc/9Shc mice on the other hand show a misregulation of sensory markers together with a hypersensitivity for cold and itch stimuli. In the sympathetic nervous system, Ret9Frs/9Frs animals display a reduced repression of cholinergic markers, with unchanged noradrenergic specification. We conclude that the studied adaptors have tissue- and cell type-specific roles and that they are main regulators of cell type specification both in the sensory and sympathetic nervous system. One central process during sympathetic nervous system development is the segregation of the noradrenergic and cholinergic lineages. While several regulating factors are known, the knowledge about how they are organized into a regulatory network is incomplete and is still missing several regulatory elements. In Paper III we investigate the gene regulatory network that controls this segregation. We show that sympathetic progenitors are a hybrid population expressing markers of both the cholinergic and noradrenergic lineage and that the homeobox transcription factor HMX1 is required both for the repression of the expression of Ret and other cholinergic markers and for the maintenance of noradrenergic marker expression. RET on the other hand maintains cholinergic marker expression and supresses HMX1

A barrel-related interneuron in layer 4 of rat somatosensory cortex with a high intra-barrel connectivity

Synaptic connections between identified fast-spiking (FS), parvalbu-min (PV)-positive interneurons, and excitatory spiny neurons in layer 4 (L4) of the barrel cortex were investigated using patch-clamp re-cordings and simultaneous biocytin fillings. Three distinct clusters of FS L4 interneurons were identified based on their axonal morphology relative to the barrel column suggesting that these neurons do not constitute a homogeneous interneuron population. One L4 FS inter-neuron type had an axonal domain strictly confined to a L4 barrel and was therefore named “barrel-confined inhibitory interneuron ” (BIn). BIns established reliable inhibitory synaptic connections with L4 spiny neurons at a high connectivity rate of 67%, of which 69 % were reci-procal. Unitary IPSPs at these connections had a mean amplitude of 0.9 ± 0.8 mV with little amplitude variation and weak short-term sy-naptic depression. We found on average 3.7 ± 1.3 putative inhibitory synaptic contacts that were not restricted to perisomatic areas. I

A Multiplex Fluidic Chip for Rapid Phenotypic Antibiotic Susceptibility Testing

Many patients with severe infections receive inappropriate empirical treatment, and rapid detection of bacterial antibiotic susceptibility can improve clinical outcome and reduce mortality. To this end, we have developed a multiplex fluidic chip for rapid phenotypic antibiotic susceptibility testing of bacteria. A total of 21 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were acquired from the EUCAST Development Laboratory and tested against amikacin, ceftazidime, and meropenem (Gram-negative bacteria) or gentamicin, ofloxacin, and tetracycline (Gram-positive bacteria). The bacterial samples were mixed with agarose and loaded in an array of growth chambers in the chip where bacterial microcolony growth was monitored over time using automated image analysis. MIC values were automatically obtained by tracking the growth rates of individual microcolonies in different regions of antibiotic gradients. Stable MIC values were obtained within 2 to 4 h, and the results showed categorical agreement with reference MIC values as determined by broth microdilution in 86% of the cases

Visceral motor neuron diversity delineates a cellular basis for nipple- and pilo-erection muscle control

Despite the variety of physiological and target-related functions, little is known regarding the cellular complexity in the sympathetic ganglion. We explored the heterogeneity of mouse stellate and thoracic ganglia and found an unexpected variety of cell types. We identified specialized populations of nipple- and pilo-erector muscle neurons. These neurons extended axonal projections and were born among other neurons during embryogenesis, but remained unspecialized until target organogenesis occurred postnatally. Target innervation and cell-type specification was coordinated by an intricate acquisition of unique combinations of growth factor receptors and the initiation of expression of concomitant ligands by the nascent erector muscles. Overall, our results provide compelling evidence for a highly sophisticated organization of the sympathetic nervous system into discrete outflow channels that project to well-defined target tissues and offer mechanistic insight into how diversity and connectivity are established during development