Dorsal-ventral Axis Research Articles

Tetrabromobisphenol A perturbs cell fate decisions via BMP signaling in the early embryonic development of zebrafish

Tetrabromobisphenol A (TBBPA) readily accumulates in the egg yolk of aquatic oviparous animals and is transferred to their embryos. Early embryogenesis is vital for organ formation and subsequent development. The developmental toxicity of TBBPA in aquatic animals has been extensively reported. However, few studies have assessed the toxic effects of TBBPA in the early embryonic development. In this work, we found that TBBPA perturbed cell fate decisions along the dorsal-ventral (DV) axis during gastrulation, further disrupting early organogenesis in the entire embryo. TBBPA exposure increased the number of embryonic cells that acquired a ventral cell fate, which formed epidermis, blood and heart tissues. In return, the number of embryonic cells that acquired a dorsal cell fate was greatly decreased, causing the TBBPA-exposed embryos to develop a small brain and small eyes. We revealed that TBBPA elevated the activity gradient of bone morphogenetic protein (BMP) signaling which is responsible for cell fate specification along the DV axis, with up-regulation of BMP ligands (bmp4, bmp7a) and target genes (szl) and promotion signal transduction through phosphorylation of Smad1/5. As the function of BMP signaling in embryogenesis is highly conserved among many vertebrates, these findings highlight the ecological and health risks of TBBPA.

Prickle isoform participation in distinct polarization events in the Drosophila eye.

Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by polarizing a cell fate choice and determining the direction and extent of ommatidial rotation. The pksple isoform of the PCP protein Prickle is known to participate in the R3/R4 cell fate decision, but the control of other polarization events and the potential contributions of the three Pk isoforms have not been clarified. Here, by characterizing expression and subcellular localization of individual isoforms together with re-analyzing isoform specific phenotypes, we show that the R3/R4 fate decision, its coordination with rotation direction, and completion of rotation to a final ±90° rotation angle are separable polarization decisions with distinct Pk isoform requirements and contributions. Both pksple and pkpk can enforce robust R3/R4 fate decisions, but only pksple can correctly orient them along the dorsal-ventral axis. In contrast, pksple and pkpk can fully and interchangeably sustain coordination of rotation direction and rotation to completion. We propose that expression dynamics and competitive interactions determine isoform participation in these processes. We propose that the selective requirement for pksple to orient the R3/R4 decision and their interchangeability for coordination and completion of rotation reflects their previously described differential interaction with the Fat/Dachsous system which is known to be required for orientation of R3/R4 decisions but not for coordination or completion of rotation.

Dorsal-ventral axis formation in sea urchin embryos.

Most sea urchin species produce planktonic feeding larvae with distinct dorsal-ventral polarity. Such morphological indicators of polarity arise after gastrulation, when several morphogenesis and cell differentiation events occur differentially along the dorsal-ventral axis. For instance, the gut bends toward the ventral side where the mouth will form, skeletogenesis occurs initially near the ventral side with the forming skeleton extending dorsally, and pigment cells differentiate and embed in the dorsal ectoderm. The patterning mechanisms and gene regulatory networks underlying these events have been extensively studied. Two opposing TGF-β signaling pathways, Nodal and BMP, play key roles in all three germ layers to respectively pattern the sea urchin ventral and dorsal sides. In this chapter, I describe our current understanding of sea urchin dorsal-ventral patterning mechanisms. Additionally, differences in the patterning mechanisms observed in lecithotrophic sea urchins (nonfeeding larvae) and in cidaroid sea urchins are also discussed, along with evolutionary insights gained from comparative analyses.

Axonal Projection Patterns of the Dorsal Interneuron Populations in the Embryonic Hindbrain.

Unraveling the inner workings of neural circuits entails understanding the cellular origin and axonal pathfinding of various neuronal groups during development. In the embryonic hindbrain, different subtypes of dorsal interneurons (dINs) evolve along the dorsal-ventral (DV) axis of rhombomeres and are imperative for the assembly of central brainstem circuits. dINs are divided into two classes, class A and class B, each containing four neuronal subgroups (dA1-4 and dB1-4) that are born in well-defined DV positions. While all interneurons belonging to class A express the transcription factor Olig3 and become excitatory, all class B interneurons express the transcription factor Lbx1 but are diverse in their excitatory or inhibitory fate. Moreover, within every class, each interneuron subtype displays its own specification genes and axonal projection patterns which are required to govern the stage-by-stage assembly of their connectivity toward their target sites. Remarkably, despite the similar genetic landmark of each dINs subgroup along the anterior-posterior (AP) axis of the hindbrain, genetic fate maps of some dA/dB neuronal subtypes uncovered their contribution to different nuclei centers in relation to their rhombomeric origin. Thus, DV and AP positional information has to be orchestrated in each dA/dB subpopulation to form distinct neuronal circuits in the hindbrain. Over the span of several decades, different axonal routes have been well-documented to dynamically emerge and grow throughout the hindbrain DV and AP positions. Yet, the genetic link between these distinct axonal bundles and their neuronal origin is not fully clear. In this study, we reviewed the available data regarding the association between the specification of early-born dorsal interneuron subpopulations in the hindbrain and their axonal circuitry development and fate, as well as the present existing knowledge on molecular effectors underlying the process of axonal growth.

Molluskan Dorsal-Ventral Patterning Relying on BMP2/4 and Chordin Provides Insights into Spiralian Development and Evolution.

Although a conserved mechanism relying on BMP2/4 and Chordin is suggested for animal dorsal–ventral (DV) patterning, this mechanism has not been reported in spiralians, one of the three major clades of bilaterians. Studies on limited spiralian representatives have suggested markedly diverse DV patterning mechanisms, a considerable number of which no longer deploy BMP signaling. Here, we showed that BMP2/4 and Chordin regulate DV patterning in the mollusk Lottia goshimai, which was predicted in spiralians but not previously reported. In the context of the diverse reports in spiralians, it conversely represents a relatively unusual case. We showed that BMP2/4 and Chordin coordinate to mediate signaling from the D-quadrant organizer to induce the DV axis, and Chordin relays the symmetry-breaking information from the organizer. Further investigations on L. goshimai embryos with impaired DV patterning suggested roles of BMP signaling in regulating the behavior of the blastopore and the organization of the nervous system. These findings provide insights into the evolution of animal DV patterning and the unique development mode of spiralians driven by the D-quadrant organizer.

Lysosomal degradation of the maternal dorsal determinant Hwa safeguards dorsal body axis formation.

The Spemann and Mangold Organizer (SMO) is of fundamental importance for dorsal ventral body axis formation during vertebrate embryogenesis. Maternal Huluwa (Hwa) has been identified as the dorsal determinant that is both necessary and sufficient for SMO formation. However, it remains unclear how Hwa is regulated. Here, we report that the E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) is essential for restricting the spatial activity of Hwa and therefore correct SMO formation in Xenopus laevis. ZNRF3 interacts with and ubiquitinates Hwa, thereby regulating its lysosomal trafficking and protein stability. Perturbation of ZNRF3 leads to the accumulation of Hwa and induction of an ectopic axis in embryos. Ectopic expression of ZNRF3 promotes Hwa degradation and dampens the axis-inducing activity of Hwa. Thus, our findings identify a substrate of ZNRF3, but also highlight the importance of the regulation of Hwa temporospatial activity in body axis formation in vertebrate embryos.

Toll-Dorsal signaling regulates the spatiotemporal dynamics of yolk granule tubulation during Drosophila cleavage

The Toll-Dorsal signaling pathway controls dorsal-ventral (DV) patterning in early Drosophila embryos, which defines specific cell fates along the DV axis and controls morphogenetic behavior of cells during gastrulation and beyond. The extent by which DV patterning information regulates subcellular organization in pre-gastrulation embryos remains unclear. We find that during Drosophila cleavage, the late endosome marker Rab7 is increasingly recruited to the yolk granules and promotes the formation of dynamic membrane tubules. The biogenesis of yolk granule tubules is positively regulated by active Rab7 and its effector complex HOPS, but negatively regulated by the Rab7 effector retromer. The occurrence of tubules is strongly biased towards the ventral side of the embryo, which we show is controlled by the Toll-Dorsal signaling pathway. Our work provides the first evidence for the formation and regulation of yolk granule tubulation in oviparous embryos and elucidates an unexpected role of Toll-Dorsal signaling in regulating this process.

Strain maps characterize the symmetry of convergence and extension patterns during zebrafish gastrulation

During gastrulation of the zebrafish embryo, the cap of blastoderm cells organizes into the axial body plan of the embryo with left–right symmetry and head–tail, dorsal–ventral polarities. Our labs have been interested in the mechanics of early development and have investigated whether these large-scale cell movements can be described as tissue-level mechanical strain by a tectonics-based approach. The first step is to image the positions of all nuclei from mid-epiboly to early segmentation by digital sheet light microscopy, organize the surface of the embryo into multi-cell spherical domains, construct velocity fields from the movements of these domains and extract strain rate maps from the change in density of the domains. During gastrulation, tensile/expansive and compressive strains in the axial and equatorial directions are detected as anterior and posterior expansion along the anterior–posterior axis and medial–lateral compression across the dorsal–ventral axis and corresponds to the well characterized morphological movements of convergence and extension. Following gastrulation strain is represented by localized medial expansion at the onset of segmentation and anterior expansion at the onset of neurulation. In addition to linear strain, symmetric patterns of rotation/curl are first detected in the animal hemispheres at mid-epiboly and then the vegetal hemispheres by the end of gastrulation. In embryos treated with C59, a Wnt inhibitor that inhibits head and tail extension, the axial extension and vegetal curl are absent. By analysing the temporal sequence of large-scale movements, deformations across the embryo can be attributed to a combination of epiboly and dorsal convergence-extension.

The Core Promoter Is a Regulatory Hub for Developmental Gene Expression.

The development of multicellular organisms and the uniqueness of each cell are achieved by distinct transcriptional programs. Multiple processes that regulate gene expression converge at the core promoter region, an 80 bp region that directs accurate transcription initiation by RNA polymerase II (Pol II). In recent years, it has become apparent that the core promoter region is not a passive DNA component, but rather an active regulatory module of transcriptional programs. Distinct core promoter compositions were demonstrated to result in different transcriptional outputs. In this mini-review, we focus on the role of the core promoter, particularly its downstream region, as the regulatory hub for developmental genes. The downstream core promoter element (DPE) was implicated in the control of evolutionarily conserved developmental gene regulatory networks (GRNs) governing body plan in both the anterior-posterior and dorsal-ventral axes. Notably, the composition of the basal transcription machinery is not universal, but rather promoter-dependent, highlighting the importance of specialized transcription complexes and their core promoter target sequences as key hubs that drive embryonic development, differentiation and morphogenesis across metazoan species. The extent of transcriptional activation by a specific enhancer is dependent on its compatibility with the relevant core promoter. The core promoter content also regulates transcription burst size. Overall, while for many years it was thought that the specificity of gene expression is primarily determined by enhancers, it is now clear that the core promoter region comprises an important regulatory module in the intricate networks of developmental gene expression.

O-157 Micro-Computed Tomography of the adult mouse ovary: an in-silico 3D reconstruction of folliculogenesis

Abstract Study question Which are the spatial dynamics of follicles recruitment and growth inside the ovary? Summary answer 3D micro-Computed Tomography (microCT) shows a simultaneous and homogeneous distribution of follicle recruitment all-over the cortex,and subsequent growth within the same ovarian region. What is known already In the mouse ovary, folliculogenesis progresses from the primordial type 1 (T1) to the fully-grown T8 follicle. Most of our knowledge of the folliculogenetic process has been obtained by disaggregating the ovary into its functional units (i.e., follicles and oocytes), thus losing the complexity of the whole histo-functional context. To date, few studies employed 3D imaging approaches to gain information on the inside 3D ovary organisation. MicroCT is the only technique that combines a high spatial resolution (down to ∼1 µm) with the production of a true 3D organ reconstruction, with cubic voxels and isotropic resolution. Study design, size, duration Three ovaries of three different adult mice were treated with the contrast agent and then imaged with microCT. A typical experiment required a total of 35 man/h from ovaries isolation to completion of X-ray scanning, and 24 man/h for follicles classification and mapping. Participants/materials, setting, methods Three ovaries of three different 8-week-old CD1 mice were fixed in 4% Paraformaldehyde and treated with Lugol’s solution for 3 hr at RT. Ovaries were scanned with Skyscan 1172 (Bruker) using a 1.5 µm/pixel resolution. MicroCT sections were processed with Fiji ImageJ (NIH), and 3D rendering of follicles and blood vessels were obtained with Avizo-9 (Thermo Fisher Scientific). ANOVA and Bonferroni post-­hocstatistical analyses were performed with RStudio, considering data significantly different when p < 0.05. Main results and the role of chance Using microCT we built the first in silico3D reconstruction of the tiny mouse ovary, identifying, mapping and counting follicles,from pre-antral secondary T4 (53.2 + 12.7 µm in diameter) to fully-grown antral T8 (321.0 + 21.3 µm), and the corpora lutea.MicroCTbrought up the main functional compartments of the growing follicle, i.e., granulosa and cumulus cells, the antrum, the zona pellucida, and the oocyte with its nucleus. Instead, primordial and primary follicles (T1–T3) could not be observed, perhaps due to the reduced size of their enclosed oocyte and to the absence of a well-formed zona pellucida around the germ cell. In addition, our analysis allowed the visualisation and 3D modelling of the main ovarian vasculature, from the largest vessel that enters the organ at the hilum site (∼150 µm size in diameter)to smaller branches present in the medulla region (∼35 µm). These results show that each of the eight ovarian sectors, virtually segmented along the dorsal-ventral axis,houses an equal number of each follicle type, suggesting a simultaneous and homogeneous distribution of follicle recruitment all-over the cortex,and subsequent growth within the same ovarian region. Limitations, reasons for caution To strengthen the results, the number of ovaries/individuals analysed should be increased. Wider implications of the findings This 3D mapping of follicles and vessels could contribute our understanding of folliculogenesis dynamics, not only under normal conditions, but also during ageing, after hormones or drugs administration, or in the presence of ovarian pathologies. Trial registration number not applicable

O-045 Synthetic human embryo-like structures: a new paradigm for human embryology

Abstract text Early human development remains mysterious and very difficult to study. Recent advances in mammalian embryology, stem cell biology, organoid technology, and bioengineering have contributed to a significant interest in bottom-up, synthetic stem cell-derived models of human development (or embryoids). The controllability and reproducibility of human embryoids coupled with the ease of genetically modifying stem cell lines, the ability to manipulate culture conditions and the simplicity of live imaging make them robust and attractive systems to disentangle cellular behaviors and signaling interactions that drive human embryogenesis. In this talk, I will describe our effort in using human pluripotent stem cells (hPSCs) to develop tractable experimental models of the peri-implantation embryonic development and neurulation. The peri-implantation human embryoids developed by us recapitulate key early post-implantation developmental landmarks successively, including pro-amniotic cavity formation, amniotic ectoderm-epiblast patterning, primordial germ cell specification, and development of the primitive streak with controlled anteroposterior polarity. I will further discuss an hPSC-based neuroectoderm patterning model to recapitulate the formation of the neural plate and another patterned neural tube model with fully defined anterior-posterior and dorsal-ventral axes.

A Characterization of the Electrophysiological and Morphological Properties of Vasoactive Intestinal Peptide (VIP) Interneurons in the Medial Entorhinal Cortex (MEC).

Circuit interactions within the medial entorhinal cortex (MEC) translate movement into a coherent code for spatial location. Entorhinal principal cells are subject to strong lateral inhibition, suggesting that a disinhibitory mechanism may drive their activation. Cortical Vasoactive Intestinal Peptide (VIP) expressing inhibitory neurons are known to contact other interneurons and excitatory cells and are thus capable of providing a local disinhibitory mechanism, yet little is known about this cell type in the MEC. To investigate the electrophysiological and morphological properties of VIP cells in the MEC, we use in vitro whole-cell patch-clamp recordings in VIPcre/tdTom mice. We report several gradients in electrophysiological properties of VIP cells that differ across laminae and along the dorsal-ventral MEC axis. We additionally show that VIP cells have distinct morphological features across laminae. Together, these results characterize the cellular and morphological properties of VIP cells in the MEC.

To Better Generate Organoids, What Can We Learn From Teratomas?

The genomic profile of animal models is not completely matched with the genomic profile of humans, and 2D cultures do not represent the cellular heterogeneity and tissue architecture found in tissues of their origin. Derived from 3D culture systems, organoids establish a crucial bridge between 2D cell cultures and in vivo animal models. Organoids have wide and promising applications in developmental research, disease modeling, drug screening, precision therapy, and regenerative medicine. However, current organoids represent only single or partial components of a tissue, which lack blood vessels, native microenvironment, communication with near tissues, and a continuous dorsal-ventral axis within 3D culture systems. Although efforts have been made to solve these problems, unfortunately, there is no ideal method. Teratoma, which has been frequently studied in pathological conditions, was recently discovered as a new in vivo model for developmental studies. In contrast to organoids, teratomas have vascularized 3D structures and regions of complex tissue-like organization. Studies have demonstrated that teratomas can be used to mimic multilineage human development, enrich specific somatic progenitor/stem cells, and even generate brain organoids. These results provide unique opportunities to promote our understanding of the vascularization and maturation of organoids. In this review, we first summarize the basic characteristics, applications, and limitations of both organoids and teratomas and further discuss the possibility that in vivo teratoma systems can be used to promote the vascularization and maturation of organoids within an in vitro 3D culture system.

Micro-electrode Recordings in Stereotactic Functional Neurosurgery

Accurate targeting to the sensorimotor territory of the globus pallidus internus (GPi), the subthalamic nucleus (STN), and the thalamic ventral intermediate nucleus (Vim) is crucial for the stereotactic neurosurgery of lesioning or implantation of a deep brain stimulation electrode. Intra-operative micro-electrode recording can aid in the localization of these structures. Neurons in the GPi are characterized by high-frequency tonic discharge. The STN has a high density of neurons that show irregular discharge. The majority of neurons in the sensorimotor territory of the GPi and STN exhibit neuronal responses to passive manipulation or active movement of the limbs and orofacial parts, and they are somatotopically arranged. Neurons responding to movements of the legs, arms, and face were found along the dorsal-ventral axis in the posterolateral GPi and along the medial-lateral axis in the posterolateral STN. Similarly, in the lateral part of the thalamic Vim nucleus, most neurons exhibit kinesthetic responses that show well-organized somatotopic arrangement, with the leg on the dorsolateral side, arm on the medioventral side, and face on the more medial side. Some Vim neurons have rhythmic bursting discharges synchronous with limb tremor. These electrophysiological findings allow us to achieve better surgical results.

Role of BMP signaling during early development of the annelid Capitella teleta

The mechanisms regulating nervous system development are still unknown for a wide variety of taxa. In insects and vertebrates, bone morphogenetic protein (BMP) signaling plays a key role in establishing the dorsal-ventral (D-V) axis and limiting the neuroectoderm to one side of that axis, leading to speculation about the conserved evolution of centralized nervous systems. Studies outside of insects and vertebrates show a more diverse picture of what, if any role, BMP signaling plays in neural development across Bilateria. This is especially true in the morphologically diverse Spiralia (≈Lophotrochozoa). Despite several studies of D-V axis formation and neural induction in spiralians, there is no consensus for how these two processes are related, or whether BMP signaling may have played an ancestral role in either process. To determine the function of BMP signaling during early development of the spiralian annelid Capitella teleta, we incubated embryos and larvae in BMP4 protein for different amounts of time. Adding exogenous BMP protein to early-cleaving C. teleta embryos had a striking effect on formation of the brain, eyes, foregut, and ventral midline in a time-dependent manner. However, adding BMP did not block brain or VNC formation or majorly disrupt the D-V axis. We identified three key time windows of BMP activity. 1) BMP treatment around birth of the 3rd-quartet micromeres caused the loss of the eyes, radialization of the brain, and a reduction of the foregut, which we interpret as a loss of A- and C-quadrant identities with a possible trans-fate switch to a D-quadrant identity. 2) Treatment after the birth of micromere 4d induced formation of a third ectopic brain lobe, eye, and foregut lobe, which we interpret as a trans-fate switch of B-quadrant micromeres to a C-quadrant identity. 3) Continuous BMP treatment from late cleavage (4d ​+ ​12 ​h) through mid-larval stages resulted in a modest expansion of Ct-chrdl expression in the dorsal ectoderm and a concomitant loss of the ventral midline (neurotroch ciliary band). Loss of the ventral midline was accompanied by a collapse of the bilaterally-symmetric ventral nerve cord, although the total amount of neural tissue was not greatly affected. Our results compared with those from other annelids and molluscs suggest that BMP signaling was not ancestrally involved in delimiting neural tissue to one region of the D-V axis. However, the effects of ectopic BMP on quadrant-identity during cleavage stages may represent a non-axial organizing signal that was present in the last common ancestor of annelids and mollusks. Furthermore, in the last common ancestor of annelids, BMP signaling may have functioned in patterning ectodermal fates along the D-V axis in the trunk. Ultimately, studies on a wider range of spiralian taxa are needed to determine the role of BMP signaling during neural induction and neural patterning in the last common ancestor of this group. Ultimately, these comparisons will give us insight into the evolutionary origins of centralized nervous systems and body plans.

Adult Hippocampal Neurogenesis and Affective Disorders: New Neurons for Psychic Well-Being.

A paradigm shift in neuroscience was the discovery that new neurons are constantly produced in the adult mammalian brain of several species, including Homo sapiens. These new-born cells are formed in some main neurogenic niches, including the subventricular zone (SVZ) at the margin of the lateral ventricle and subgranular zone (SGZ) in the hippocampal dentate gyrus (DG). In the DG, neuroblasts derive from SGZ progenitors and migrate to the hippocampal granular layer becoming adult granule cells, which are integrated into functional adult circuits. It has been confirmed that adult hippocampal neurogenesis (AHN) is a long-lasting phenomenon in the human brain. The functions of hippocampal new-born cells are not fully established. Experimental studies suggest that they have unique electrophysiological properties, including hyperexcitability, which enable them to regulate adult granule cells. Their specific function depends on the anatomical hippocampal location along the hippocampal dorsal-ventral axis. Dorsal hippocampus plays a more defined role on spatial learning and contextual information, while the ventral hippocampus is more related to emotional behavior, stress resilience and social interaction. Several reports suggest a role for AHN in pattern separation, cognitive flexibility, forgetting and reversal learning. It has been proposed that deficits in AHN might impair normal DG function, including pattern separation and cognitive flexibility, which could play a role on the etiology of affective disorders, such as depression, anxiety and post-traumatic stress disorder (PTSD). In this paper, we review recent scientific evidence suggesting that impairment of AHN may underlie the pathophysiology of affective disorders even in humans and that neurogenesis-inspired therapies may be a promising approach to reduce symptoms of affective disorders in humans.

Evolutionary Conservation of Hox Genes in Vertebrate Brain Development

Hox genes, their conserved derivatives, and the pathways responsible for their expression have been extensively studied in the fruit fly, Drosophila melanogaster;the experimentation done in the Drosophila model system has given developmental biologists tools to better understand the role and significance of Hox genes and their derivatives in anterior-posterior axis determination in the Drosophila embryo. Along with this, Drosophila research opened up the door to investigation on the conservation of Hox genes between vertebrates and invertebrates. Comparative embryology in mice, chickens, pufferfish, and zebrafish have shown conserved Hox gene expression patterns specifically along the anterior-posterior axis. Recently, comparative analysis performed on dorsal-ventral axis formation showed that patterning and segmentation of the spinal cord is influenced by the action of Hox genes as well. This review will briefly consider the evolution of the vertebrate brain and the evolution and conservation of Hox genes in regulating hindbrain patterning and spinal cord development.

A reaction-diffusion network model predicts a dual role of Cactus/IκB to regulate Dorsal/NFκB nuclear translocation in Drosophila.

Dorsal-ventral patterning of the Drosophila embryo depends on the NFκB superfamily transcription factor Dorsal (Dl). Toll receptor activation signals for degradation of the IκB inhibitor Cactus (Cact), leading to a ventral-to-dorsal nuclear Dl gradient. Cact is critical for Dl nuclear import, as it binds to and prevents Dl from entering the nuclei. Quantitative analysis of cact mutants revealed an additional Cact function to promote Dl nuclear translocation in ventral regions of the embryo. To investigate this dual Cact role, we developed a predictive model based on a reaction-diffusion regulatory network. This network distinguishes non-uniform Toll-dependent Dl nuclear import and Cact degradation, from the Toll-independent processes of Cact degradation and reversible nuclear-cytoplasmic Dl flow. In addition, it incorporates translational control of Cact levels by Dl. Our model successfully reproduces wild-type data and emulates the Dl nuclear gradient in mutant dl and cact allelic combinations. Our results indicate that the dual role of Cact depends on the dynamics of Dl-Cact trimers along the dorsal-ventral axis: In the absence of Toll activation, free Dl-Cact trimers retain Dl in the cytoplasm, limiting the flow of Dl into the nucleus; in ventral-lateral regions, Dl-Cact trimers are recruited by Toll activation into predominant signaling complexes and promote Dl nuclear translocation. Simulations suggest that the balance between Toll-dependent and Toll-independent processes are key to this dynamics and reproduce the full assortment of Cact effects. Considering the high evolutionary conservation of these pathways, our analysis should contribute to understanding NFκB/c-Rel activation in other contexts such as in the vertebrate immune system and disease.

High and low expression of the hyperpolarization activated current (Ih ) in mouse CA1 stratum oriens interneurons.

Inhibitory interneurons are among the most diverse cell types in the brain; the hippocampus itself contains more than 28 different inhibitory interneurons. Interneurons are typically classified using a combination of physiological, morphological, and biochemical observations. One broad separator is action potential firing: low threshold, regular spiking versus higher threshold, fast spiking. We found that spike frequency adaptation (SFA) was highly heterogeneous in low threshold interneurons in the mouse stratum oriens region of area CA1. Analysis with a k‐means clustering algorithm parsed the data set into two distinct clusters based on a constellation of physiological parameters and reliably sorted strong and weak SFA cells into different groups. Interneurons with strong SFA fired fewer action potentials across a range of current inputs and had lower input resistance compared to cells with weak SFA. Strong SFA cells also had higher sag and rebound in response to hyperpolarizing current injections. Morphological analysis shows no difference between the two cell types and the cell types did not segregate along the dorsal–ventral axis of the hippocampus. Strong and weak SFA cells were labeled in hippocampal slices from SST:cre Ai14 mice suggesting both cells express somatostatin. Voltage‐clamp recordings showed hyperpolarization activated current I h was significantly larger in strong SFA cells compared to weak SFA cells. We suggest that the strong SFA cell represents a previously uncharacterized type of CA1 stratum oriens interneuron. Due to the combination of physiological parameters of these cells, we will refer to them as Low Threshold High I h (LTH) cells.

A Highly Conserved Shh Enhancer Coordinates Hypothalamic and Craniofacial Development.

Enhancers that are conserved deep in evolutionary time regulate characteristics held in common across taxonomic classes. Here, deletion of the highly conserved Shh enhancer SBE2 (Shh brain enhancer 2) in mouse markedly reduced Shh expression within the embryonic brain specifically in the rostral diencephalon; however, no abnormal anatomical phenotype was observed. Secondary enhancer activity was subsequently identified which likely mediates low levels of expression. In contrast, when crossing the SBE2 deletion with the Shh null allele, brain and craniofacial development were disrupted; thus, linking SBE2 regulated Shh expression to multiple defects and further enabling the study of the effects of differing levels of Shh on embryogenesis. Development of the hypothalamus, derived from the rostral diencephalon, was disrupted along both the anterior-posterior (AP) and the dorsal-ventral (DV) axes. Expression of DV patterning genes and subsequent neuronal population induction were particularly sensitive to Shh expression levels, demonstrating a novel morphogenic context for Shh. The role of SBE2, which is highlighted by DV gene expression, is to step-up expression of Shh above the minimal activity of the second enhancer, ensuring the necessary levels of Shh in a regional-specific manner. We also show that low Shh levels in the diencephalon disrupted neighbouring craniofacial development, including mediolateral patterning of the bones along the cranial floor and viscerocranium. Thus, SBE2 contributes to hypothalamic morphogenesis and ensures there is coordination with the formation of the adjacent midline cranial bones that subsequently protect the neural tissue.