Lhx1 is required in Müllerian duct epithelium for uterine development.

The female reproductive tract organs of mammals, including the oviducts, uterus, cervix and upper vagina, are derived from the Müllerian ducts, a pair of epithelial tubes that form within the mesonephroi. The Müllerian ducts form in a rostral to caudal manner, guided by and dependent on the Wolffian ducts that have already formed. Experimental embryological studies indicate that caudal elongation of the Müllerian duct towards the urogenital sinus occurs in part by proliferation at the ductal tip. The molecular mechanisms that regulate the elongation of the Müllerian duct are currently unclear. Lhx1 encodes a LIM-homeodomain transcription factor that is essential for male and female reproductive tract development. Lhx1 is expressed in both the Wolffian and Müllerian ducts. Wolffian duct-specific knockout of Lhx1 results in degeneration of the Wolffian duct and consequently the non-cell-autonomous loss of the Müllerian duct. To determine the role of Lhx1 specifically in the Müllerian duct epithelium, we performed a Müllerian duct-specific knockout study using Wnt7a-Cre mice. Loss of Lhx1 in the Müllerian duct epithelium led to a block in Müllerian duct elongation and uterine hypoplasia characterized by loss of the entire endometrium (luminal and glandular epithelium and stroma) and inner circular but not the outer longitudinal muscle layer. Time-lapse imaging and molecular analyses indicate that Lhx1 acts cell autonomously to maintain ductal progenitor cells for Müllerian duct elongation. These studies identify LHX1 as the first transcription factor that is essential in the Müllerian duct epithelial progenitor cells for female reproductive tract development. Furthermore, these genetic studies demonstrate the requirement of epithelial-mesenchymal interactions for uterine tissue compartment differentiation.

Transient receptor potential A1 activation prolongs isoflurane induction latency and impairs respiratory function in mice.

BACKGROUND: Isoflurane is a potent volatile anesthetic; however, it evokes airway irritation and neurogenic constriction through transient receptor potential (TRP) A1 channels and sensitizes TRPV1 channels, which colocalizes with TRPA1 in most of the vagal C-fibers innervating the airway. However, little is known about the precise effects of these two channels on the respiratory function during isoflurane anesthesia. METHODS: By using a rodent behavioral model and whole-body plethysmograph, the authors examined the response of Trpa1 and Trpv1 mice to isoflurane anesthesia and monitored their respiratory functions during anesthesia. RESULTS: This study showed that Trpa1 mice (n = 9), but not Trpv1 mice (n = 11), displayed a shortened induction latency compared with wild-type mice (n = 10) during isoflurane anesthesia (33 ± 2.0 s in wild-type and 33 ± 3.8 s in Trpv1 vs. 17 ± 1.8 in Trpa1 at 2.2 minimum alveolar concentrations). By contrast, their response to the nonpungent volatile anesthetic sevoflurane is indistinguishable from wild-type mice (24 ± 3.6 s in wild-type vs. 26 ± 1.0 s in Trpa1 at 2.4 minimum alveolar concentrations). The authors discovered that Trpa1 mice inhaled more anesthetic but maintained better respiratory function. Further respiration pattern analysis revealed that isoflurane triggered nociceptive reflexes and led to prolonged resting time between breaths during isoflurane induction as well as decreased dynamic pulmonary compliance, an indicator of airway constriction, throughout isoflurane anesthesia in wild-type and Trpv1 mice, but not in Trpa1 mice. CONCLUSION: Activation of TRPA1 by isoflurane negatively affects anesthetic induction latency by altering respiratory patterns and impairing pulmonary compliance.

Pain and itch coding mechanisms of polymodal sensory neurons.

Pain and itch coding mechanisms in polymodal sensory neurons remain elusive. MrgprD+ neurons represent a major polymodal population and mediate both mechanical pain and nonhistaminergic itch. Here, we show that chemogenetic activation of MrgprD+ neurons elicited both pain- and itch-related behavior in a dose-dependent manner, revealing an unanticipated compatibility between pain and itch in polymodal neurons. While VGlut2-dependent glutamate release is required for both pain and itch transmission from MrgprD+ neurons, the neuropeptide neuromedin B (NMB) is selectively required for itch signaling. Electrophysiological recordings further demonstrated that glutamate synergizes with NMB to excite NMB-sensitive postsynaptic neurons. Ablation of these spinal neurons selectively abolished itch signals from MrgprD+ neurons, without affecting pain signals, suggesting a dedicated itch-processing central circuit. These findings reveal distinct neurotransmitters and neural circuit requirements for pain and itch signaling from MrgprD+ polymodal sensory neurons, providing new insights on coding and processing of pain and itch.

A mouse reporter line to conditionally mark nuclei and cell membranes for in vivo live-imaging.

Live-imaging is an essential tool to visualize live cells and monitor their behaviors during development. This technology demands a variety of mouse reporter lines, each uniquely expressing a fluorescent protein. Here, we developed an R26R-RG reporter mouse line that conditionally and simultaneously expresses mCherry and EGFP in nuclei and plasma membranes, respectively, from the Rosa26 locus. The intensity and resolution of mCherry nuclear localization and EGFP membrane localization were demonstrated to be sufficient for live-imaging with embryos that express RG (mCherry and EGFP) ubiquitously and specifically in fetal Sertoli cells. The conditional R26R-RG reporter mouse line should be a useful tool for labeling nuclei and membranes simultaneously in distinct cell populations.

A cerebellar-thalamocortical pathway drives behavioral context-dependent movement initiation.

Executing learned motor behaviors often requires the transformation of sensory cues into patterns of motor commands that generate appropriately timed actions. The cerebellum and thalamus are two key areas involved in shaping cortical output and movement, but the contribution of a cerebellar-thalamocortical pathway to voluntary movement initiation remains poorly understood. Here, we investigated how an auditory "go cue" transforms thalamocortical activity patterns and how these changes relate to movement initiation. Population responses in dentate/interpositus-recipient regions of motor thalamus reflect a time-locked increase in activity immediately prior to movement initiation that is temporally uncoupled from the go cue, indicative of a fixed-latency feedforward motor timing signal. Blocking cerebellar or motor thalamic output suppresses movement initiation, while stimulation triggers movements in a behavioral context-dependent manner. Our findings show how cerebellar output, via the thalamus, shapes cortical activity patterns necessary for learned context-dependent movement initiation.

Electroporation of Postimplantation Mouse Embryos In Utero.

Gene transfer by electroporation is possible in mouse fetuses within the uterus. As described in this protocol, the pregnant female is anesthetized, the abdominal cavity is opened, and the uterus with the fetuses is exteriorized. A solution of plasmid DNA is injected through the uterine wall directly into the fetus, typically into a cavity like the brain ventricle, guided by fiber optic illumination. Electrodes are positioned on the uterus around the region of the fetus that was injected, and electrical pulses are delivered. The uterus is returned to the abdominal cavity, the body wall is sutured closed, and the female is allowed to recover. The manipulated fetuses can then be collected and analyzed at various times after the electroporation. This method allows experimental access to later-stage developing mouse embryos.

Anatomical and functional dichotomy of ocular itch and pain.

Itch and pain are refractory symptoms of many ocular conditions. Ocular itch is generated mainly in the conjunctiva and is absent from the cornea. In contrast, most ocular pain arises from the cornea. However, the underlying mechanisms remain unknown. Using genetic axonal tracing approaches, we discover distinct sensory innervation patterns between the conjunctiva and cornea. Further genetic and functional analyses in rodent models show that a subset of conjunctival-selective sensory fibers marked by MrgprA3 expression, rather than corneal sensory fibers, mediates ocular itch. Importantly, the actions of both histamine and nonhistamine pruritogens converge onto this unique subset of conjunctiva sensory fibers and enable them to play a key role in mediating itch associated with allergic conjunctivitis. This is distinct from skin itch, in which discrete populations of sensory neurons cooperate to carry itch. Finally, we provide proof of concept that selective silencing of conjunctiva itch-sensing fibers by pruritogen-mediated entry of sodium channel blocker QX-314 is a feasible therapeutic strategy to treat ocular itch in mice. Itch-sensing fibers also innervate the human conjunctiva and allow pharmacological silencing using QX-314. Our results cast new light on the neural mechanisms of ocular itch and open a new avenue for developing therapeutic strategies.

A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons.

Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations. VIDEO ABSTRACT.

Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells.

Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control. DOI:http://dx.doi.org/10.7554/eLife.00400.001.

Stromal-to-epithelial transition during postpartum endometrial regeneration.

Endometrium is the inner lining of the uterus which is composed of epithelial and stromal tissue compartments enclosed by the two smooth muscle layers of the myometrium. In women, much of the endometrium is shed and regenerated each month during the menstrual cycle. Endometrial regeneration also occurs after parturition. The cellular mechanisms that regulate endometrial regeneration are still poorly understood. Using genetic fate-mapping in the mouse, we found that the epithelial compartment of the endometrium maintains its epithelial identity during the estrous cycle and postpartum regeneration. However, whereas the stromal compartment maintains its identity during homeostatic cycling, after parturition a subset of stromal cells differentiates into epithelium that is subsequently maintained. These findings identify potential progenitor cells within the endometrial stromal compartment that produce long-term epithelial tissue during postpartum endometrial regeneration.

Mice harboring Gnrhr E90K, a mutation that causes protein misfolding and hypogonadotropic hypogonadism in humans, exhibit testis size reduction and ovulation failure.

GnRH, produced in the hypothalamus, acts on pituitary gonadotropes to stimulate release of the gonadotropins LH and FSH. Reduced responsiveness of gonadotropes to GnRH is a primary cause of hypogonadotropic hypogonadism (HH), a disease characterized by gonadal dysfunction and low blood levels of gonadotropins. Loss-of-function mutations in the gene encoding the receptor for GnRH (GNRHR) are a common cause of HH. Sequencing of the GNRHR gene in patients with HH revealed mainly point mutations producing single amino acid substitutions that cause misfolding and misrouting of this G protein-coupled receptor. To generate a mouse model that mimics the human disease, we introduced a single amino acid substitution (E90K) into the mouse Gnrhr gene, which is identical to a known human recessive mutation. In humans, E90K causes severe HH by preventing formation of the E90-K121 salt bridge, which is essential for correct folding. In cell cultures, E90K causes misfolding that leads to almost complete retention by the protein quality control system and subsequent degradation. Here we report that the primary phenotype of mice homozygous for E90K is female infertility due to ovulation failure. Mutant males are fertile despite reduced gonadotropin levels and smaller testes. These results suggest decreased GnRH receptor signaling in the mutant animal, compared with wild type. Our findings suggest that a threshold level of GnRH receptor activity is required for ovulation.