Jennifer M. Skidmore

Characterization of Progenitor Domains in the Developing Mouse Thalamus

To understand the molecular basis of the specification of thalamic nuclei, we analyzed the expression patterns of various transcription factors and defined progenitor cell populations in the embryonic mouse thalamus. We show that the basic helix‐loop‐helix (bHLH) transcription factor Olig3 is expressed in the entire thalamic ventricular zone and the zona limitans intrathalamica (ZLI). Next, we define two distinct progenitor domains within the thalamus, which we name pTH‐R and pTH‐C, located caudal to the ZLI. pTH‐R is immediately caudal to the ZLI and expresses Nkx2.2, Mash1, and Olig3. pTH‐C is caudal to pTH‐R and expresses Ngn1, Ngn2, and Olig3. Short‐term lineage analysis of Olig3‐, Mash1‐, Ngn1‐, and Ngn2‐expressing progenitor cells as well as tracing the Pitx2 cell lineage suggests that pTH‐C is the only major source of thalamic nuclei containing neurons that project to the cerebral cortex, whereas pTH‐R and ZLI are likely to produce distinct postmitotic populations outside of the cortex‐projecting part of the thalamus. To determine if pTH‐C is composed of subdomains, we characterized expression of the homeodomain protein Dbx1 and the bHLH protein Olig2. We show that Dbx1 is expressed in caudodorsal‐high to rostroventral‐low gradient within pTH‐C. Analysis of heterozygous Dbx1nlslacZ knockin mice demonstrated that Dbx1‐expressing progenitors preferentially give rise to caudodorsal thalamic nuclei. Olig2 is expressed in an opposite gradient within pTH‐C to that of Dbx1. These results establish the molecular heterogeneity within the progenitor cells of the thalamus, and suggest that such heterogeneity contributes to the specification of thalamic nuclei. J. Comp. Neurol. 505:73–91, 2007.

Molecular basis for defective glycosylation and Pseudomonas pathogenesis in cystic fibrosis lung

The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P. aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments.

Polar clustering of the chemoreceptor complex in Escherichia coli occurs in the absence of complete CheA function.

Bacterial chemotaxis requires a phosphorelay system initiated by the interaction of a ligand with its chemoreceptor and culminating in a change in the directional bias of flagellar rotation. Chemoreceptor-CheA-CheW ternary complexes mediate transduction of the chemotactic signal. In vivo, these complexes cluster predominantly in large groups at the cell poles. The function of chemoreceptor clustering is currently unknown. To gain insight into the relationship between signaling and chemoreceptor clustering, we examined these properties in several Escherichia coli mutant strains that produce CheA variants altered in their ability to mediate chemotaxis, autophosphorylate, or bind ATP. We show here that polar clustering of chemoreceptor complexes does not require functional CheA protein, although maximal clustering occurred only in chemotactically competent cells. Surprisingly, in cells containing a minimum of 13 gold particles at the cell pole, a significant level of clustering was observed in the absence of CheA, demonstrating that CheA is not absolutely essential for chemoreceptor clustering. Nonchemotactic cells expressing only CheA(S), a C-terminal CheA deletion, or CheA bearing a mutation in the ATP-binding site mediated slightly less than maximal chemoreceptor clustering. Cells expressing only full-length CheA (CheA(L)) from either a chromosomal or a plasmid-encoded allele displayed a methyl-accepting chemotaxis protein localization pattern indistinguishable from that of strains carrying both CheA(L) and CheA(S), demonstrating that CheA(L) alone can mediate polar clustering.

Localization and environmental regulation of MCP‐like proteins in Rhodobacter sphaeroides

Chemotaxis to many compounds by Rhodobacter sphaeroides requires transport and at least partial metabolism of the chemoeffector. Previous investigations using phototrophically grown cells have failed to find any homologues of the MCP chemoreceptors identified in Escherichia coli. However, using an antibody raised against the highly conserved domain of E. coli Tsr, MCP‐like proteins were identified in R. sphaeroides WS8N. Analysis using Western blotting and immunogold electron microscopy showed that expression of these MCP‐like proteins is environmentally regulated and that receptors are targeted to two different cellular locations: the poles of the cells and the cytoplasm. In aerobically grown cells, these proteins were shown by immunoelectron microscopy to localize predominantly to the cell poles and to an electron‐dense body in the cytoplasm. Western blot analysis indicated a 17‐fold reduction in protein concentration when cells were grown in the light. The number of immunogold particles was also dramatically reduced in anaerobically light‐grown cells and their cellular distribution was altered. Fewer receptors localized to the cell poles and more particles randomly distributed within the cell, but the cytoplasmic cluster remained. These trends were more pronounced in cells grown anaerobically under dim light than in those grown anaerobically under bright light, suggesting that expression is controlled by redox state and either light intensity or the extent of photosynthetic membrane synthesis. Recent work on E. coli chemosensing suggests that oligomerization of receptors and chemosensory proteins is important for sensory signalling. The data presented here suggest that this oligomerization can occur with cytoplasmic receptors and also provides an explanation for the multiple copies of chemosensory proteins in R. sphaeroides.

The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels

The Obg subfamily of bacterial GTP‐binding proteins are biochemically distinct from Ras‐like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtAC) which, in Ras‐like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP‐binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtAC, we generated a temperature‐sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtAC is important for 50S assembly and/or stability. Surprisingly, at the non‐permissive temperature, G80E  cells  rapidly  lose  viability  and  yet  do not display an additional ribosome defect. Thus, the essential nature of the cgtAC gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1‐S arrest. Therefore, CgtAC is necessary for DNA replication and progression through the cell cycle.

Regulators of membrane trafficking and Mycobacterium tuberculosis phagosome maturation block.

The biogenesis and maturation of phagosomes is an area of study which has been employing aspects of proteomic analyses and variations on that theme by identifying components on isolated organelles and following their dynamic changes and interactions with the endocytic pathway. In the case of Mycobacterium tuberculosis phagosome, the arrest of its maturation in infected macrophages, referred to in classical texts as the inhibition of phagosome‐lysosome fusion, represents a phenomenon that is used to functionally dissect the phagosomal maturation pathway. In this review, we summarize the recent studies on regulators of membrane trafficking and other organelle components in the context of phagosomal biogenesis and mycobacterial phagosome maturation arrest.

CHD7 and retinoic acid signaling cooperate to regulate neural stem cell and inner ear development in mouse models of CHARGE syndrome

CHARGE syndrome is a multiple congenital anomaly disorder that leads to life-threatening birth defects, such as choanal atresia and cardiac malformations as well as multiple sensory impairments, that affect hearing, vision, olfaction and balance. CHARGE is caused by heterozygous mutations in CHD7, which encodes an ATP-dependent chromatin remodeling enzyme. Identification of the mechanisms underlying neurological and sensory defects in CHARGE is a first step toward developing treatments for CHARGE individuals. Here, we used mouse models of Chd7 deficiency to explore the function of CHD7 in the development of the subventricular zone (SVZ) neural stem cell niche and inner ear, structures that are important for olfactory bulb neurogenesis and hearing and balance, respectively. We found that loss of Chd7 results in cell-autonomous proliferative, neurogenic and self-renewal defects in the perinatal and mature mouse SVZ stem cell niche. Modulation of retinoic acid (RA) signaling prevented in vivo inner ear and in vitro neural stem cell defects caused by Chd7 deficiency. Our findings demonstrate critical, cooperative roles for RA and CHD7 in SVZ neural stem cell function and inner ear development, suggesting that altered RA signaling may be an effective method for treating Chd7 deficiency.

Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function

The Caulobacter crescentus CgtA protein is a member of the Obg/GTP1 subfamily of monomeric GTP‐binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP and displays rapid exchange rate constants for either nucleotide, indicating that the guanine nucleotide‐binding and exchange properties of CgtA are different from those of the well‐characterized Ras‐like GTP‐binding proteins. The Obg/GTP1 proteins share sequence similarity along the putative effector‐binding domain. In this study, we examined the functional consequences of altering amino acid residues within this conserved domain, and identified that T193 was critical for CgtA function. The in vitro binding, exchange and GTP hydrolysis of the T192A, T193A and T192AT193A mutant proteins was examined using fluorescent guanine nucleotide analogues (mant‐GDP and mant‐GTP). Substitution of either T192 and/or T193 for alanine modestly reduced binding to GDP and significantly reduced the binding affinity for GTP. Furthermore, the T193A mutant protein was more severely impaired for binding GTP than the T192A mutant. The T193A mutation appeared to account solely for the impaired GTP binding of the T192AT193A double mutation. This is the first report that demonstrates that a confirmed defect in guanine nucleotide binding and GTP hydrolysis of an Obg‐like protein results in the lack of function in vivo.

Cre fate mapping reveals lineage specific defects in neuronal migration with loss of Pitx2 function in the developing mouse hypothalamus and subthalamic nucleus.

Establishment of neuronal diversity is a central topic in developmental neurobiology. Prior studies implicated Pitx2, a paired-like homeodomain transcription factor, in mouse subthalamic nucleus neuronal development, but precise stages of neuronal differentiation affected (migration, axon outgrowth, fate specification) and underlying mechanisms were unknown. Here we report lineage tracing experiments using Pitx2(cre/+), Pitx2(cre/null), and conditional nuclear lacZ reporter mice to track embryonic Pitx2 expressing neurons. Migration of subthalamic nucleus and hypothalamic neurons was severely arrested in Pitx2(cre/null) embryos, and subclasses of subthalamic nucleus neurons identified by Lmx1b, Foxp1, and Foxp2-gene expression revealed differing sensitivities to Pitx2 dosage. Interestingly, embryonic subthalamic nucleus development was unaffected in Lmx1b null mice, suggesting that Pitx2 and Lmx1b act via independent genetic pathways. These data provide the first direct evidence for Pitx2-dependent neuronal migration in the developing hypothalamus, and demonstrate that complex transcriptional networks regulate regional specialization of distinct hypothalamic and subthalamic nucleus neurons.

GABAergic and glutamatergic identities of developing midbrain Pitx2 neurons

Pitx2, a paired‐like homeodomain transcription factor, is expressed in post‐mitotic neurons within highly restricted domains of the embryonic mouse brain. Previous reports identified critical roles for PITX2 in histogenesis of the hypothalamus and midbrain, but the cellular identities of PITX2‐positive neurons in these regions were not fully explored. This study characterizes Pitx2 expression with respect to midbrain transcription factor and neurotransmitter phenotypes in mid‐to‐late mouse gestation. In the dorsal midbrain, we identified Pitx2‐positive neurons in the stratum griseum intermedium (SGI) as GABAergic and observed a requirement for PITX2 in GABAergic differentiation. We also identified two Pitx2‐positive neuronal populations in the ventral midbrain, the red nucleus, and a ventromedial population, both of which contain glutamatergic precursors. Our data suggest that PITX2 is present in regionally restricted subpopulations of midbrain neurons and may have unique functions that promote GABAergic and glutamatergic differentiation. Developmental Dynamics 240:333–346, 2011.