Probes for CRE

The central nucleus of the amygdala (CeA) is a key hub integrating sensory inputs and modulating behavioural outputs. The CeA is a complex structure with discrete subdivisions, high peptidergic heterogeneity and broad CNS afferent and efferent projections. While several neuropeptide systems within the CeA have been examined in detail, less is known about CeA preproenkephalin (ppENK) cells. Here, we used a recently developed transgenic Penk-Cre mouse line to advance our understanding of the efferent and afferent connectivity of ppENK in the CeA. First, to determine the fidelity of Cre expression in Penk-Cre transgenic mice, we conducted RNAscope in the CeA of Penk-Cre mice. Our analysis revealed that 96.6% of CeA Cre+ neurons co-expressed pENK mRNA, and 99.7% of CeA pENK+ neurons co-expressed Cre mRNA, indicating faithful recapitulation of Cre expression in CeA ppENK-expressing cells, supporting the fidelity of the Penk-Cre reporter mouse. Anterograde tracing of CeAPenk cells showed strong efferent projections to the extended amygdala, midbrain and hindbrain PBN and NTS. Retrograde tracing of Penk afferents to the CeA were more restricted, with primary innervation originating within the amygdala complex and bed nucleus of the stria terminalis, and minor innervation from the parabrachial nucleus and nucleus of the solitary tract. Together, our data provide a comprehensive map of ENKergic efferent and afferent connectivity of the CeA in Penk-Cre mice. Further, we highlight both the utility and limitations of the Penk-Cre mice to study the function of CeA, PBN and NTS ppENK cells.

All mammalian uteri contain glands in their endometrium that develop only or primarily after birth. In mice, those endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful for the study of uterine gland function during pregnancy. Utilizing CRISPR-Cas9 genome editing, Cre recombinase was inserted into the endogenous serine protease 29 precursor (Prss29) gene. Both Prss29 mRNA and Cre recombinase activity was specific to the GE of the mouse uterus following implantation, but was absent from other areas of the female reproductive tract. Next, Prss29-Cre mice were crossed with floxed forkhead box A2 (Foxa2) mice to conditionally delete Foxa2 specifically in the endometrial glands. Foxa2 was absent in the glands of the post-implantation uterus, and Foxa2 deleted mice exhibited complete infertility after their first pregnancy. These results establish that Prss29-Cre mice are a valuable resource to elucidate and explore the functions of glands in the adult uterus.

The neuropeptide oxytocin (Oxt) plays important roles in modulating social behaviors. Oxt receptor (Oxtr) is abundantly expressed in the brain and its relationship to socio-behavioral controls has been extensively studied using mouse brains. Several genetic tools to visualize and/or manipulate Oxtr-expressing cells, such as fluorescent reporters and Cre recombinase drivers, have been generated by ES-cell based gene targeting or bacterial artificial chromosome (BAC) transgenesis. However, these mouse lines displayed some differences in their Oxtr expression profiles probably because of the complex context and integrity of their genomic configurations in each line. Here, we apply our sophisticated genome-editing techniques to the Oxtr locus, systematically generating a series of knock-in mouse lines, in which its endogenous transcriptional regulations are intactly preserved and evaluate their expression profiles to ensure the reliability of our new tools. We employ the epitope tagging strategy, with which C-terminally fused tags can be detected by highly specific antibodies, to successfully visualize the Oxtr protein distribution on the neural membrane with super-resolution imaging for the first time. By using T2A self-cleaving peptide sequences, we also induce proper expressions of tdTomato reporter, codon-improved Cre recombinase (iCre), and spatiotemporally inducible Cre-ERT2 in Oxtr-expressing neurons. Electrophysiological recordings from tdTomato-positive cells in the reporter mice support the validity of our tool design. Retro-orbital injections of AAV-PHP.eB vector into the Cre line further enabled visualization of recombinase activities in the appropriate brain regions. Moreover, the first-time Cre-ERT2 line drives Cre-mediated recombination in a spatiotemporally controlled manner on tamoxifen (TMX) administration. These tools thus provide an excellent resource for future functional studies in Oxt-responsive neurons and should prove of broad interest in the field.

The cortex of the adrenal gland is organized into concentric zones that produce distinct steroid hormones essential for body homeostasis in mammals. Mechanisms leading to the development, zonation and maintenance of the adrenal cortex are complex and have been studied since the 1800s. However, the advent of genetic manipulation and transgenic mouse models over the past 30 years has revolutionized our understanding of these mechanisms. This review lists and details the distinct Cre recombinase mouse strains available to study the adrenal cortex, and the remarkable progress total and conditional knockout mouse models have enabled us to make in our understanding of the molecular mechanisms regulating the development and maintenance of the adrenal cortex.

The renin-angiotensin system (RAS) has been implicated in stress-related disorders, however the central mechanisms responsible for this remains unknown. The locus coeruleus (LC), a major noradrenergic nucleus of the brain, plays a critical role in modulating anxiety-like behaviors. The LC has also been previously shown to express angiotensinogen (AGT), the pre-cursor for angiotensin, as well as strong expression of angiotensin II receptors, but its role in stress-related disorders has not been examined. Using angiotensin II type 1 receptor (AT1R)-eGFP and Cre mice combined with neuroanatomical and behavioral approaches, we examined the role of LC expressing AT1R in fear- and anxiety-related behavior.

Early-life trauma (ELT) is a risk factor for binge eating and obesity later in life, yet the neural circuits that underlie this association have not been addressed. Here, we show in mice that downregulation of the leptin receptor (Lepr) in the lateral hypothalamus (LH) and its effect on neural activity is crucial in causing ELT-induced binge-like eating and obesity upon high-fat diet exposure. We also found that the increased activity of Lepr-expressing LH (LHLepr) neurons encodes sustained binge-like eating in ELT mice. Inhibition of LHLepr neurons projecting to the ventrolateral periaqueductal gray normalizes these behavioral features of ELT mice. Furthermore, activation of proenkephalin-expressing ventrolateral periaqueductal gray neurons, which receive inhibitory inputs from LHLepr neurons, rescues ELT-induced maladaptive eating habits. Our results identify a circuit pathway that mediates ELT-induced maladaptive eating and may lead to the identification of novel therapeutic targets for binge eating and obesity.

Obesity is a global pandemic that is causally linked to many life-threatening diseases. Apart from some rare genetic conditions, the biological drivers of overeating and reduced activity are unclear. Here, we show that neurotensin-expressing neurons in the mouse interstitial nucleus of the posterior limb of the anterior commissure (IPAC), a nucleus of the central extended amygdala, encode dietary preference for unhealthy energy-dense foods. Optogenetic activation of IPACNts neurons promotes obesogenic behaviors, such as hedonic eating, and modulates food preference. Conversely, acute inhibition of IPACNts neurons reduces feeding and decreases hedonic eating. Chronic inactivation of IPACNts neurons recapitulates these effects, reduces preference for sweet, non-caloric tastants and, furthermore, enhances locomotion and energy expenditure; as a result, mice display long-term weight loss and improved metabolic health and are protected from obesity. Thus, the activity of a single neuronal population bidirectionally regulates energy homeostasis. Our findings could lead to new therapeutic strategies to prevent and treat obesity.

The chromatin organization of the gonadotropin gene promoters in the pituitary gonadotropes plays a major role in determining how these gene are activated, but is difficult to study because of the low numbers of these cells in the pituitary gland. Here, we set out to create a cell model to study gonadotropin chromatin, and found that by optimizing cell culture conditions, we can maintain stable proliferating cultures of primary non-transformed gonadotrope cells over weeks to months. Although expression of the gonadotropin genes drops very low, these cells are enriched in gonadotrope markers and respond to GnRH. Furthermore, >85% of the cells contained Lhb and/or Fshb mature transcripts; though these were virtually restricted to the nuclei. The gonadotropes were harvested initially due to expression of dTOMATO, following activation of Cre recombinase by the Gnrhr promoter. Over 6 mo in culture, a similar proportion of the recombined DNA was maintained (i.e. cells derived from the original gonadotropes or having acquired Gnrhr-promoter activity), together with cells of a distinct origin. The cells are enriched with markers of proliferating pituitary and stem cells, including Sox2, suggesting that multipotent precursor cells might have proliferated and differentiated into gonadotrope-like cells. These cell cultures offer a new and versatile methodology for research in gonadotrope differentiation and function, and can provide enough primary cells for chromatin immunoprecipitation and epigenetic analysis, while our initial studies also indicate a possible regulatory mechanism that might be involved in the nuclear export of gonadotropin gene mRNAs.

The sinoatrial node (SAN) functions as the pacemaker of the heart, initiating rhythmic heartbeats. Despite its importance, the SAN is one of the most poorly understood cardiac entities because of its small size and complex composition and function. The Hippo signaling pathway is a molecular signaling pathway fundamental to heart development and regeneration. Although abnormalities of the Hippo pathway are associated with cardiac arrhythmias in human patients, the role of this pathway in the SAN is unknown.We investigated key regulators of the Hippo pathway in SAN pacemaker cells by conditionally inactivating the Hippo signaling kinases Lats1 and Lats2 using the tamoxifen-inducible, cardiac conduction system-specific Cre driver Hcn4CreERT2 with Lats1 and Lats2 conditional knockout alleles. In addition, the Hippo-signaling effectors Yap and Taz were conditionally inactivated in the SAN. To determine the function of Hippo signaling in the SAN and other cardiac conduction system components, we conducted a series of physiological and molecular experiments, including telemetry ECG recording, echocardiography, Masson Trichrome staining, calcium imaging, immunostaining, RNAscope, cleavage under targets and tagmentation sequencing using antibodies against Yap1 or H3K4me3, quantitative real-time polymerase chain reaction, and Western blotting. We also performed comprehensive bioinformatics analyses of various datasets.We found that Lats1/2 inactivation caused severe sinus node dysfunction. Compared with the controls, Lats1/2 conditional knockout mutants exhibited dysregulated calcium handling and increased fibrosis in the SAN, indicating that Lats1/2 function through both cell-autonomous and non-cell-autonomous mechanisms. It is notable that the Lats1/2 conditional knockout phenotype was rescued by genetic deletion of Yap and Taz in the cardiac conduction system. These rescued mice had normal sinus rhythm and reduced fibrosis of the SAN, indicating that Lats1/2 function through Yap and Taz. Cleavage Under Targets and Tagmentation sequencing data showed that Yap potentially regulates genes critical for calcium homeostasis such as Ryr2 and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as Tgfb1 and Tgfb3. Consistent with this, Lats1/2 conditional knockout mutants had decreased Ryr2 expression and increased Tgfb1 and Tgfb3 expression compared with control mice.We reveal, for the first time to our knowledge, that the canonical Hippo-Yap pathway plays a pivotal role in maintaining SAN homeostasis.

Reactive oxygen species (ROS) produced by NADPH oxidases (Nox) contribute to the development of different types of sensorineural hearing loss (SNHL), a common impairment in humans with no established treatment. Although the essential role of Nox3 in otoconia biosynthesis and its possible involvement in hearing have been reported in rodents, immunohistological methods targeted at detecting Nox3 expression in inner ear cells reveal ambiguous results. Therefore, the mechanism underlying Nox3-dependent SNHL remains unclear and warrants further investigation. We generated Nox3-Cre knock-in mice, in which Nox3 was replaced with Cre recombinase (Cre). Using Nox3-Cre;tdTomato mice of either sex, in which tdTomato is expressed under the control of the Nox3 promoter, we determined Nox3-expressing regions and cell types in the inner ear. Nox3-expressing cells in the cochlea included various types of supporting cells (SC), outer hair cells (OHC), inner hair cells (IHC), and spiral ganglion neurons (SGN). Nox3 expression increased with cisplatin, age, and noise insults. Moreover, increased Nox3 expression in SC and OHC, especially at the basal turn of the cochlea, played essential roles in ROS-related SNHL. The extent of Nox3 involvement in SNHL follows the following order: cisplatin-induced HL (CIHL) > age-related HL (ARHL) > noise-induced HL (NIHL). Here, on the basis of Nox3-Cre;tdTomato, which can be used as a reporter system (Nox3-Cre+/-;tdTomato+/+ and Nox3-Cre+/+;tdTomato+/+ ), and Nox3-KO (Nox3-Cre+/+;tdTomato+/+ ) mice, we demonstrate that Nox3 inhibition in the cochlea is a promising strategy for ROS-related SNHL, such as CIHL, ARHL, and NIHL.SIGNIFICANCE STATEMENT:We found Nox3-expressing regions and cell-types in the inner ear, especially in the cochlea, using Nox3-Cre;tdTomato mice, a reporter system generated in this study. Nox3 expression increased with cisplatin, age, and noise insults in specific cell-types in the cochlea and resulted in the loss (apoptosis) of outer hair cells. Thus, Nox3 might serve as a molecular target for the development of therapeutics for sensorineural hearing loss, particularly cisplatin-induced, age-related, and noise-induced hearing loss.

The Temporomandibular joint (TMJ) is one of the most complex joints in the human body. TMJ is composed of the temporal bone, a disc and a movable mandibular condyle with abundant tendon attachments. Tendon has been thought to play the sole function of transmitting muscle forces to stabilize joints, yet it is largely unclear why tendon undergoes ectopic ossification in trauma or diseases, and whether it has any direct contribution to skeletal formation. This study aimed to investigate the full biological significance of tendon in TMJ growth. We first discovered that the TMJ condyle is composed of a well-established cartilage head and an overlooked “bony head” that grows after birth and continuously expands throughout the lifespan with little signs of remodeling. Mouse X-ray images (Fig.1a) showed little change in the cartilage head’s volume but a continuous expansion in the bony head’s mass with a low mineral content from 1 to 5 months (Fig.1b). Toluidine blue staining showed TMJ condyle had a large area of tendon attachment extending down to ramus (Fig.1c, white dotted line in lower magnification), defined by regions of tendon, interface, and TFB (Fig.1c1). The TFB morphology was distinct from endosteum-formed bone (EFB, Fig.1c1), cartilage-formed bone (CFB, Fig.1c2, rich in cartilage residual), or periosteum-formed bone (PFB, Fig.1c3) in cell shape and distribution, and ECM. TEM images further revealed that the osteocytes in the TFB were large in size, irregular in shape, had small nuclei but numerous ERs and Golgi complexes, and were embedded in ECM rich in fibropositors. In contrast, the osteocytes in EFB, CFB or PFB were spindle-shaped with larger nuclei but fewer ERs and Golgi complexes (Fig.1d). To reveal the cell source of the bony head, cell lineage tracing were used. Tracing data showed that most CFB cells originate from Col10a1+ hypertrophic chondrocytes, whereas the interface and TFB were derived from Scx+ cells (Fig.1e). RNAscope displayed high levels of Thbs4 (Thrombospondin-4, a tendon marker) and SOST (a potent inhibitor of Wnt signaling secreted by osteocytes) mRNA in TFB at bony head (Fig.1f). The Scx-CreERT2 tracing combined with IHC staining showed TFB maintained a mixed ECM of bone (Col1), cartilage (Aggrecan) and tendon (Periostin, Fig.1g). To further determine the role of tendon lineage in condyle expansion, we generated Scx-CreERT2; R26RDTA (carrying a loxP-flanked stop cassette associated with an attenuated diphtheria toxin fragment A, DTA, for the ablation of cells when Cre is active). Deletion of Scx+ cells greatly reduced the size of bony head (Fig.1h) and the thickness of interface with few Scx+/Col1+ bone cells in P28 DTA mice (Fig.1i); In conclusion, our study tendon cells, beyond their conventional role in joint movement, are key players for the postnatal growth and expansion of TMJ condyle (Fig.1j).