The Egf-Ras-Erk Pathway and the Nkx-5/hmx Homeodomain Protein Mls-2 Promote Tube Development in the C.elegans Excretory System

Tubular epithelial cells are one of the most abundant cell types in multicellular organisms. Tubular cells transport gases and liquids, and funnel harmful excretory waste from our bodies. It is clear that Receptor Tyrosine Kinase (RTK) signaling is essential for the formation of many tubular organs such as our kidneys and blood vessels. However, which steps in tube development require RTK signaling is less well understood. The C.elegans excretory system is a primitive renal system with just three essential cells (duct, pore, and canal cells), providing a simple yet dynamic system to study tube specification and morphogenesis. In the C.elegans excretory system, we demonstrated that the EGF-Ras-Erk signaling pathway specified the excretory duct tube versus the pore tube fate. In addition, EGF-Ras-Erk signaling influenced the positions that the duct and pore cells adopted within the tubular network. And finally, after position and fate determination, EGF-Ras-Erk signaling had a continued role in maintaining organ architecture of the duct tube. Goals for future research will be to determine how EGF-Ras-ERK signaling controls these genetically distinct steps during tube development. In a separate project, I studied the Nkx5 homeodomain transcription factor, MLS-2, which was identified in a mutagenesis screen by a former graduate student in the lab. I discovered a role for MLS-2 in promoting proper cell shape of the duct and pore. mls-2 cooperated with the EGF-Ras-Erk pathway to turn on lin-48/Ovo during duct morphogenesis. I speculate that MLS-2 and other Nkx5 family members have conserved functions in promoting shape acquisition in cells that adopt complex morphologies similar to the duct and pore

Beyond somatosensation: Mrgprs in mucosal tissues

Mas-related G coupled receptors (Mrgprs) are a superfamily of receptors expressed in sensory neurons that are known to transmit somatic sensations from the skin to the central nervous system. Interestingly, Mrgprs have recently been implicated in sensory and motor functions of mucosal-associated neuronal circuits. The gastrointestinal and pulmonary tracts are constantly exposed to noxious stimuli. Therefore, it is likely that neuronal Mrgpr signaling pathways in mucosal tissues, akin to their family members expressed in the skin, might relay messages that alert the host when mucosal tissues are affected by damaging signals. Further, Mrgprs have been proposed to mediate the cross-talk between sensory neurons and immune cells that promotes host-protective functions at barrier sites. Although the mechanisms by which Mrgprs are activated in mucosal tissues are not completely understood, these exciting studies implicate Mrgprs as potential therapeutic targets for conditions affecting the intestinal and airway mucosa. This review will highlight the central role of Mrgpr signaling pathways in the regulation of homeostasis at mucosal tissues

Evoked and Spontaneous Pain Assessment During Tooth Pulp Injury

Injury of the tooth pulp is excruciatingly painful and yet the receptors and neural circuit mechanisms that transmit this form of pain remain poorly defined in both the clinic and preclinical rodent models. Easily quantifiable behavioral assessment in the mouse orofacial area remains a major bottleneck in uncovering molecular mechanisms that govern inflammatory pain in the tooth. In this study we sought to address this problem using the Mouse Grimace Scale and a novel approach to the application of mechanical Von Frey hair stimuli. We use a dental pulp injury model that exposes the pulp to the outside environment, a procedure we have previously shown produces inflammation. Using RNAscope technology, we demonstrate an upregulation of genes that contribute to the pain state in the trigeminal ganglia of injured mice. We found that mice with dental pulp injury have greater Mouse Grimace Scores than sham within 24 hours of injury, suggestive of spontaneous pain. We developed a scoring system of mouse refusal to determine thresholds for mechanical stimulation of the face with Von Frey filaments. This method revealed that mice with a unilateral dental injury develop bilateral mechanical allodynia that is delayed relative to the onset of spontaneous pain. This work demonstrates that tooth pain can be quantified in freely behaving mice using approaches common for other types of pain assessment. Harnessing these assays in the orofacial area during gene manipulation should assist in uncovering mechanisms for tooth pulp inflammatory pain and other forms of trigeminal pain. © 2020, The Author(s)

Functional interrogation of an odorant receptor locus reveals multiple axes of transcriptional regulation.

The odorant receptor (OR) genes constitute the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion, through an unknown mechanism that likely exploits positive and negative regulation. We devised a genetic strategy in mice to examine OR selection by determining the transcriptional activity of an exogenous promoter homologously integrated into an OR locus. Using the tetracycline-dependent transactivator responsive promoter (tet(o)), we observed that the OR locus imposes spatial and temporal constraints on tet(o)-driven transcription. Conditional expression experiments reveal a developmental change in the permissiveness of the locus. Further, expression of an OR transgene that suppresses endogenous ORs similarly represses the OR-integrated tet(o). Neurons homozygous for the tet(o)-modified allele demonstrate predominantly monoallelic expression, despite their potential to express both copies. These data reveal multiple axes of regulation, and support a model of initiation of OR choice limited by nonpermissive chromatin and maintained by repression of nonselected alleles

Frequency and zonal restriction of tTa-driven tet_o-modified P2 alleles in the olfactory epithelium.

<p>(A) Diagram of the olfactory epithelium showing zones of OR expression. The shaded region is the II/III zone of P2 expression. Areas in black boxes depict regions shown in (F–N). (B) Coronal section through the olfactory epithelium of a P2-IRES-GFP control animal reveals expression of the P2 allele at P14. Sections were subject to anti-GFP IHC (green), and nuclei were counterstained with Toto-3 (blue). (C) Coronal section through the olfactory epithelium of a OMP-IRES-tTa/tet-P2-IRES-GFP animal reveals expression of the tet-P2 allele driven by tTa at P14. Sections were subject to anti-GFP IHC (green), and nuclei were counterstained with Toto-3 (blue). (D) High-power image of the boxed region in (B). (E) High-power image of the boxed region in (C). (F–H) Coronal sections through the olfactory epithelium of a CaMKII-tTa/tet-P2-IRES-GFP animal reveal the zonal restriction of expression of the tet-P2 allele driven by tTa at P75 in zone I/II (F), zone II/III (G), and zone IV (H). Sections were subject to anti-GFP IHC (green), and nuclei were counterstained with Toto-3 (blue). (I–K) Coronal sections through the olfactory epithelium of a CaMKII-tTa/tet-P2Δ-IRES-GFP animal reveal the zonal restriction of expression of the tet-P2 allele driven by tTa at P75 in zone I/II (I), zone II/III (J), and zone IV (K). Sections were subject to anti-GFP IHC (green), and nuclei were counterstained with Toto-3 (blue). (L–N) Coronal sections through the olfactory epithelium of a CaMKII-tTa/M71-Tg animal show pervasive expression of tet-linked M71 transgene driven by tTa at P60 in zone I/II (L), zone II/III (M), and zone IV (N). Sections were subject to anti-lacZ IHC (green), and nuclei were counterstained with Toto-3 (blue). (O–T) Zonal restriction of the tet-P2 allele driven by OMP-IRES-tTa examined by two-color RNA in situ hybridization. Coronal sections through olfactory epithelia of P90 OMP-IRES-tTa/tet-P2 animals were hybridized with RNA probes directed against GFP (green) (O and R), and against OMP (red) (P and S), in zonal region II/III (O–Q) and zonal region IV (R–T). Red and green channels are shown merged (Q and T). Nuclei were counterstained with Toto-3 (blue). (U–Z) Increase in frequency of expression of the tet-P2 allele over time. Coronal sections corresponding to zone II/III of the olfactory epithelia of OMP-IRES-tTa/tet-P2 animals subject to IHC with immunoserum directed against GFP (green) at P14 (U), P18 (V), P30 (W), P60 (X), P120 (Y), and P360 (Z). Nuclei counterstained with Toto-3 (blue).</p

Construction of the tet-P2Z allele and predominant allelic exclusion of the homozygous tet_o-modified P2 alleles.

<p>(A) Modification of the endogenous P2 locus by homologous recombination to generate the tet-P2Z allele. (I) The tet-P2Z targeting construct allows bicistronic expression of the P2 OR protein and the marker protein tau-lacZ, both driven by the tet operator inserted at the start site of transcription of the P2 locus. Flanking P2 promoter regions are preserved in the construct, shifted 5′ of the tet operator. (II) The unmodified genomic P2 locus. (III) Homologous recombination in mouse ES cells followed by self-excision of the ACN selection cassette yields the tet-P2Z allele. (B) Diagram of the genetic strategy used to for biallelic expression of the tet_o-modified P2 alleles in the mouse olfactory epithelium in vivo. The tet-P2 and tet-P2Z alleles have the potential to be transcribed in all olfactory sensory neurons of the olfactory epithelium by the ubiquitous expression of tTa from the CaMKII-tTa transgene. (C and D) Expression of the tet-P2Z allele in the olfactory epithelium (C) and the VNO (D) revealed by IHC in coronal sections with antibody directed against lacZ (red) in a CaMKII-tTa/tet-P2Z animal. Nuclei are revealed by Toto-3 counterstain. (E–G) Expression of the tet-P2 and tet-P2Z alleles in a compound heterozygous animal CaMKII-tTa/tet-P2/tet-P2Z shown by immunohistochemical detection of GFP (green) (E) and lacZ (red) (F), and with merged signals (G). Nuclei are revealed by Toto-3 counterstaining. (E′–G′) High-power magnification of a region of the fields shown in panels (E–G), respectively. An olfactory neuron exhibiting biallelic expression of the tet-P2 alleles is shown by the arrows. (H) Distribution of single (purple) and double (orange) tet-P2+ cells in olfactory epithelia of CaMKII-tTa/tet-P2/tet-P2Z animals. The mean relative position, normalized to the height of the epithelium, of single tet-P2+ cells was 0.501 and of double tet-P2+ cells was 0.424 (<i>n</i> = 100, <i>p</i><0.0068, unpaired <i>t</i>-test, two-tailed).</p

Suppression of tet_o-modified P2 alleles by the pervasive expression of an OR transgene.

<p>(A–C) Coronal sections through the olfactory epithelium of an OMP-IRES-tTa/tet-P2 mouse subject to immunohistochemical detection of lacZ (red) (A) and GFP (green) (B), and with merged signals (C). Nuclei (blue) revealed by Toto-3 counterstaining. (D–F) Coronal sections through the olfactory epithelium of an OMP-IRES-tTa/tet-P2/tet-M71 animal subject to immunohistochemical detection of lacZ (red) (D) and GFP (green) (E), and with merged signals (F). Nuclei (blue) revealed by Toto-3 counterstaining. (G–I) Coronal sections through the olfactory epithelium of an OMP-IRES-tTa/tet-P2Δ mouse subject to immunohistochemical detection of lacZ (red) (G) and GFP (green) (H), and with merged signals (I). Nuclei (blue) revealed by Toto-3 counterstaining. (J–L) Coronal sections through the olfactory epithelium of an OMP-IRES-tTa/tet-P2Δ/tet-M71 mouse subject to immunohistochemical detection of lacZ (red) (J) and GFP (green) (K), and with merged signals (L). Nuclei (blue) revealed by Toto-3 counterstaining.</p

Pre-activation of tet-P2 leads to persistent expression independent of tTa.

<p>(A) Diagram of pre-activation strategy of tet-P2 with tTa by administration of doxycycline (dox). The tet-P2 locus is subject to activation by tTa until P60 by CaMKII-tTa. Doxycycline is administered, to ablate tTa binding, for 48 h prior to expression analysis by RNA in situ hybridization. (B) Diagram of the tet-P2 allele showing the location of RNA probes used to differentiate between wild-type (red “+1”) and tet_o (black “+1”) start sites of transcription. The RNA probe shown in red is derived from tet_o sequences and detects message initiated by the endogenous P2 promoter, while the probe shown in green is derived from GFP sequences and hybridizes to messages initiated from either endogenous P2 or tet_o promoters. (C–E) Control experiments demonstrate expression of the tet-P2 gene initiated from the wild-type P2 promoter. Coronal sections of a tet-P2 animal subject to RNA in situ hybridization with probe directed against the tet_o sequences (red) (C), with probe directed against GFP sequences (green) (D), and with red and green channels merged (E). Nuclei were counterstained with Toto-3 (blue). (F–H) Expression of the tet-P2 allele driven by CaMKII-tTa without doxycycline treatment at P60. Coronal sections of a tet-P2 CaMKII-tTa animal subject to RNA in situ hybridization with probe directed against the tet_o sequences (red) (F), with probe directed against GFP sequences (green) (G), and with red and green channels merged (H). Nuclei were counterstained by Toto-3 (blue). (I–K) Continuation of expression of the tet-P2 allele driven by CaMKII-tTa after 48 h of doxycycline treatment at P60. Coronal sections of a tet-P2 CaMKII-tTa animal subject to RNA in situ hybridization with probe directed against the tet_o sequences (red) (I), with probe directed against GFP sequences (green) (J), and with red and green channels merged (K). Nuclei were counterstained with Toto-3 (blue).</p

Opposing, spatially-determined epigenetic forces impose restrictions on stochastic olfactory receptor choice

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in the mouse genome in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this ‘privileged’ repertoire. Our experiments identify early transcription as a potential ‘epigenetic’ contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression