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Cleft lip, which results from impaired facial process growth and fusion, is one of the most common cranio-facial birth defects. Many genes are known to be involved in the etiology of this disorder; however, our understanding of cleft lip pathogenesis remains incomplete. In the present study, we uncovered a role for sonic hedgehog (SHH) signaling during lip fusion. Mice carrying compound mutations in hedgehog acyltransferase (Hhat) and patched1 (Ptch1) exhibited perturbations in the SHH gradient during frontonasal development, which led to hypoplastic nasal process outgrowth, epithelial seam persistence, and cleft lip. Further investigation revealed that enhanced SHH signaling restricts canonical WNT signaling in the lambdoidal region by promoting expression of genes encoding WNT inhibitors. Moreover, reduction of canonical WNT signaling perturbed p63/interferon regulatory factor 6 (p63/IRF6) signaling, resulting in increased proliferation and decreased cell death, which was followed by persistence of the epithelial seam and cleft lip. Consistent with our results, mutations in genes that disrupt SHH and WNT signaling have been identified in both mice and humans with cleft lip. Collectively, our data illustrate that altered SHH signaling contributes to the etiology and pathogenesis of cleft lip through antagonistic interactions with other gene regulatory networks, including the canonical WNT and p63/IRF6 signaling pathways., Introduction Craniofacial development depends on the proper growth and fusion of distinct facial processes during embryogenesis. Perturbation of either developmental event can lead to facial cleft phenotypes (1, 2). It [...]

Background: The vertebrate retina is an organized laminar structure comprised of distinct cell types populating three nuclear layers. During development, each retinal cell type follows a stereotypical temporal order of genesis, differentiation, and migration, giving rise to its stratified organization. Once born, the precise positioning of cells along the apico‐basal (radial) axis of the retina is critical for subsequent connections to form, relying on highly orchestrated migratory processes. While these processes are critical for visual function to arise, the regulators of cellular migration and retinal lamination remain largely unexplored. Results: We report a role for a microtubule‐interacting protein, Mllt11 (myeloid/lymphoid or mixed‐lineage leukemia; translocated to chromosome 11/All1 fused gene from chromosome 1q) in mammalian retinal cell migration during retinogenesis. We show that Mllt11 loss‐of‐function in mouse retinal neuroblasts affected the migration of ganglion and amacrine cells into the ganglion cell layer and led to their aberrant accumulation in the inner nuclear and plexiform layers. Conclusions: We demonstrate a role for Mllt11 in neuroblast migration and formation of the ganglion cell layer of the retina. Key Findings: Mllt11 plays a role in mammalian retinal lamination during development.Mllt11 regulates neuroblast morphology important for migration into the ganglion cell layer.Mllt11 loss of function leads to aberrant accumulation of retinal ganglion cells and displaced amacrine cells in the inner plexiform layer. [ABSTRACT FROM AUTHOR]

The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are two types of cortical projection neurons (CPNs): those that project locally and interhemispherically and those that project to subcerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here, we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during mouse brain formation. We show that Mllt11 expression is exclusive to developing neurons and is enriched in the developing cortical plate (CP) during the formation of the superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gainof- function analysis we show that Mllt11 is required for neuritogenesis and proper migration of upper layer CPNs. Loss of Mllt11 in the superficial cortex of male and female neonates leads to a severe reduction in fibers crossing the corpus callosum (CC), a progressive loss in the maintenance of upper layer projection neuron gene expression, and reduced complexity of dendritic arborization. Proteomic analysis revealed that Mllt11 associates with stabilized microtubules, and Mllt11 loss affected microtubule staining in callosal axons. Taken together, our findings support a role for Mllt11 in promoting the formation of mature upper-layer neuron morphologies and connectivity in the cerebral cortex. [ABSTRACT FROM AUTHOR]

Retinoids (vitamin A and its derivatives) play essential roles during vertebrate development. Vitamin A deprivation leads to severe congenital malformations affecting many tissues, including diverse neural crest cell populations and the heart. The vitamin A signal is transduced by the retinoic acid receptors (RAR [alpha], RAR [beta], and RAR [gamma]). However, these receptors exhibit considerable functional redundancy, as judged by the mild phenotype of RAR single null mutants relative to the defects evoked by loss of multiple RARs. To circumvent this redundancy, the endogenous RAR [gamma] 2 allele was replaced with a ligand-binding RAR [gamma] mutant (RAR [gamma] [E.sub.305]) by gene targeting in mouse embryonic stem (ES) cells. Chimeric embryos derived from hemizygous RAR [gamma] [E.sub.305] ES cells displayed several defects similar to those observed in certain RAR double null mutants, including hypoplasia or absence of the caudal pharyngeal arches and myocardial deficiencies. The latter defects were not due to abnormal cardiac specification as affected hearts still expressed chamber-specific markers in an appropriate manner. Chimeras also displayed cardiac looping anomalies, which were associated with a reduction of Pitx2. This work suggests a role for RAR signaling in late looping morphogenesis and illustrates the utility of using a dominant-negative gene substitution approach to circumvent the functional redundancy inherent to the RAR family. Key Words: retinoic acid receptor; dominant negative; lateral plate mesoderm; myocardium; left-right; Pitx2; looping; chimera.

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation. [ABSTRACT FROM AUTHOR]

The cerebellum coordinates vestibular input into the hindbrain to control balance and movement, and its anatomical complexity is increasingly viewed as a high‐throughput processing center for sensory and cognitive functions. Cerebellum development however is relatively simple, and arises from a specialized structure in the anterior hindbrain called the rhombic lip, which along with the ventricular zone of the rostral‐most dorsal hindbrain region, give rise to the distinct cell types that constitute the cerebellum. Granule cells, being the most numerous cell types, arise from the rhombic lip and form a dense and distinct layer of the cerebellar cortex. In this short review, we describe the various strategies used by amniotes and anamniotes to generate and diversify granule cell types during cerebellar development. Key Findings: Strategies used by vertebrates to generate cerebellar granule cells.Role of Atoh1, Neurod1, and Cux2 in the patterning and migration of granule cell precursors. [ABSTRACT FROM AUTHOR]

Background: Vertebrate spinal cord development requires Sonic Hedgehog (Shh) signaling from the floorplate and notochord, where it is thought to act in concentration dependent manner to pattern distinct cell identities along the ventral-to-dorsal axis. While in vitro experiments demonstrate naïve neural tissues are sensitive to small changes in Shh levels, genetic studies illustrate that some degree of ventral patterning can occur despite significant perturbations in Shh signaling. Consequently, the mechanistic relationship between Shh morphogen levels and acquisition of distinct cell identities remains unclear. Results: We addressed this using Hedgehog acetyltransferase ( HhatCreface) and Wiggable mouse mutants. Hhat encodes a palmitoylase required for the secretion of Hedgehog proteins and formation of the Shh gradient. In its absence, the spinal cord develops without floorplate cells and V3 interneurons. Wiggable is an allele of the Shh receptor Patched1 (Ptch1Wig) that is unable to inhibit Shh signal transduction, resulting in expanded ventral progenitor domains. Surprisingly, HhatCreface/Creface; Ptch1Wig/Wig double mutants displayed fully restored ventral patterning despite an absence of Shh secretion from the floorplate. Conclusions: The full range of neuronal progenitor types can be generated in the absence of a Shh gradient provided pathway repression is dampened, illustrating the complexity of morphogen dynamics in vertebrate patterning. Developmental Dynamics 247:170-184, 2018. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: The rhombic lip (RL), a germinal zone in the developing hindbrain, gives rise to all of the excitatory neurons of the cerebellum. It is presently unclear what factors distinguish between RL progenitor pools and play a role in differentiating the multiple cell types that arise from this region. The transcription factor Cux2 has been shown to play important roles in proliferation and differentiation of distinct neuronal populations during embryogenesis, but its role in cerebellar fate restriction is unknown. Results: Through expression analysis and genetic fate mapping studies we show that Cux2 is expressed in the RL of the fetal brain and is restricted to a pool of cerebellar granule cell precursors and unipolar brush cells. This restriction was remarkably specific because regardless of the timing of Cux2 reporter gene activation in the RL, only granule cell layer derivatives were labeled. However, the overexpression of Cux2 in naïve hindbrain tissue was insufficient to force progenitor cells to adopt a granule cell fate. Conclusions: Our results suggest that Cux2 delineates the pool of cerebellar granule cell layer progenitors from other RL and ventricular zone derivatives, and plays a role in fate restricting, but not differentiating, this population. Developmental Dynamics 245:881-896, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Cranial nerves govern sensory and motor information exchange between the brain and tissues of the head and neck. The cranial nerves are derived from two specialized populations of cells, cranial neural crest cells and ectodermal placode cells. Defects in either cell type can result in cranial nerve developmental defects. Although several signaling pathways are known to regulate cranial nerve formation our understanding of how intercellular signaling between neural crest cells and placode cells is coordinated during cranial ganglia morphogenesis is poorly understood. Sonic Hedgehog (Shh) signaling is one key pathway that regulates multiple aspects of craniofacial development, but whether it co-ordinates cranial neural crest cell and placodal cell interactions during cranial ganglia formation remains unclear. In this study we examined a new Patched1 (Ptch1) loss-of-function mouse mutant and characterized the role of Ptch1 in regulating Shh signaling during cranial ganglia development. Ptch1Wig/ Wig mutants exhibit elevated Shh signaling in concert with disorganization of the trigeminal and facial nerves. Importantly, we discovered that enhanced Shh signaling suppressed canonical Wnt signaling in the cranial nerve region. This critically affected the survival and migration of cranial neural crest cells and the development of placodal cells as well as the integration between neural crest and placodes. Collectively, our findings highlight a novel and critical role for Shh signaling in cranial nerve development via the cross regulation of canonical Wnt signaling. [ABSTRACT FROM AUTHOR]

ABSTRACT The hippocampus arises from the medial region of the subventricular (SVZ) within the telencephalon. It is one of two regions in the postnatal brain that harbors neural progenitors (NPs) capable of giving rise to new neurons. Neurogenesis in the hippocampus is restricted to the subgranular zone (SGZ) of the dentate gyrus (DG) where it contributes to the generation of granule cell layer (gcl) neurons. It is thought that SGZ progenitors are heterogeneous, differing in their morphology, expression profiles, and developmental potential, however it is currently unknown whether they display differences in their developmental origins and cell fate-restriction in the DG. Here we demonstrate that Cux2 is a marker for SGZ progenitors and nascent granule cell neurons in the perinatal brain. Cux2 was expressed in the presumptive hippocampal forming region of the embryonic forebrain from E14.5 onwards. At fetal stages, Cux2 was expressed in early-forming Prox1+ granule cell neurons as well as the SVZ of the DG germinal matrix. In the postnatal brain, Cux2 was expressed in several types of progenitors in the SGZ of the DG, including Nestin/Sox2 double-positive radial glia, Sox2+ cells that lacked a radial glial process, DCX+ neuroblasts, and Calretinin-expressing nascent neurons. Another domain characterized by a low level of Cux2 expression emerged in Calbindin+ neurons of the developing DG blades. We used Cux2-Cre mice in genetic fate-mapping studies and showed almost exclusive labeling of Calbindin-positive gcl neurons, but not in any progenitor cell types or astroglia. This suggests that Cux2+ progenitors directly differentiate into gcl neurons and do not self-renew. Interestingly, developmental profiling of cell fate revealed an outside-in formation of gcl neurons in the DG, likely reflecting the activity of Cux2 in the germinative matrices during DG formation and maturation. However, DG morphogenesis proceeded largely normally in hypomorphic Cux2 mutants lacking Cux2 expression. Taken together we conclude that Cux2 expression reflects hippocampal neurogenesis and identifies non-self-renewing NPs in the SGZ. © 2014 The Authors Hippocampus Published by Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Holoprosencephaly (HPE) is a failure of the forebrain to bifurcate and is the most common structural malformation of the embryonic brain. Mutations in SHH underlie most familial (17%) cases of HPE; and, consistent with this, Shh is expressed in midline embryonic cells and tissues and their derivatives that are affected in HPE. It has long been recognized that a graded series of facial anomalies occurs within the clinical spectrum of HPE, as HPE is often found in patients together with other malformations such as acrania, anencephaly, and agnathia. However, it is not known if these phenotypes arise through a common etiology and pathogenesis. Here we demonstrate for the first time using mouse models that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which mimics the severe condition observed in humans. Hhat is required for post-translational palmitoylation of Hedgehog (Hh) proteins; and, in the absence of Hhat, Hh secretion from producing cells is diminished. We show through downregulation of the Hh receptor Ptch1 that loss of Hhat perturbs long-range Hh signaling, which in turn disrupts Fgf, Bmp and Erk signaling. Collectively, this leads to abnormal patterning and extensive apoptosis within the craniofacial primordial, together with defects in cartilage and bone differentiation. Therefore our work shows that Hhat loss-of-function underscrores HPE; but more importantly it provides a mechanism for the co-occurrence of acrania, holoprosencephaly, and agnathia. Future genetic studies should include HHAT as a potential candidate in the etiology and pathogenesis of HPE and its associated disorders. [ABSTRACT FROM AUTHOR]

Obtaining the diversity of interneuron subtypes in their appropriate numbers requires the orchestrated integration of progenitor proliferation with the regulation of differentiation. Here we demonstrate through loss-of-function studies in mice that the Cut homeodomain transcription factor Cux2 (Cutl2) plays an important role in regulating the formation of dorsal spinal cord interneurons. Furthermore, we show that Notch regulates Cux2 expression. Although Notch signaling can be inhibitory to the expression of proneural genes, it is also required for interneuron formation during spinal cord development. Our findings suggest that Cux2 might mediate some of the effects of Notch signaling on interneuron formation. Together with the requirement for Cux2 in cell cycle progression, our work highlights the mechanistic complexity in balancing neural progenitor maintenance and differentiation during spinal cord neurogenesis. [ABSTRACT FROM AUTHOR]

Neurogenesis requires the coordination of neural progenitor proliferation and differentiation with cell-cycle regulation. However, the mechanisms coordinating these distinct cellular activities are poorly understood. Here we demonstrate for the first time that a Cut-like homeodomain transcription factor family member, Cux2 (Cutl2), regulates cell-cycle progression and development of neural progenitors. Cux2 loss-of-function mouse mutants exhibit smaller spinal cords with deficits in neural progenitor development as well as in neuroblast and interneuron differentiation. These defects correlate with reduced cell-cycle progression of neural progenitors coupled with diminished Neurod and p27Kip1 activity. Conversely, in Cux2 gain-of-function transgenic mice, the spinal cord is enlarged in association with enhanced neuroblast formation and neuronal differentiation, particularly with respect to interneurons. Furthermore, Cux2 overexpression induces high levels of Neurod and p27Kip1. Mechanistically, we discovered through chromatin immunoprecipitation assays that Cux2 binds both the Neurod and p27Kip1 promoters in vivo, indicating that these interactions are direct. Our results therefore show that Cux2 functions at multiple levels during spinal cord neurogenesis. Cux2 initially influences cell-cycle progression in neural progenitors but subsequently makes additional inputs through Neurod and p27Kip1 to regulate neuroblast formation, cell-cycle exit and cell-fate determination. Thus our work defines novel roles for Cux2 as a transcription factor that integrates cell-cycle progression with neural progenitor development during spinal cord neurogenesis. [ABSTRACT FROM AUTHOR]

The dorsal medial region of the developing mammalian telencephalon plays a central role in the patterning of the adjacent brain regions. This review describes the development of this specialized region of the vertebrate brain, called the cortical hem , and the formation of the various cells and structures it gives rise to, including the choroid plexus, Cajal-Retzius cells and the hippocampus. We highlight the ontogenic processes that create these different forebrain derivatives from their shared embryonic origin and discuss the key signalling pathways and molecules that influence the patterning of the cortical hem. These include BMP, Wnt, FGF and Shh signalling pathways acting with Homeobox factors to carve the medial telencephalon into district progenitor regions, which in turn give rise to the choroid plexus, dentate gyrus and hippocampus. We then link the formation of the lateral ventricle choroid plexus with embryonic and postnatal neurogenesis in the hippocampus.

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Sectioning with a vibrating microtome (vibratome) is a valuable procedure for generating thick sections that can be used for immunohistochemistry and in situ hybridization. It is particularly useful for revealing histological and 3D detail in tissues and embryos that have been subjected to various whole-mount histological procedures such as β-galactosidase and alkaline phosphatase staining, and fluorescent and DAB (diaminobenzidine) immunostaining. Vibratome sectioning does not involve any harsh organic solvents and is therefore suited for processing specimens stained with fluorescent antibodies or dyes. In addition, live tissue can be sectioned for subsequent culture, manipulation, and live imaging. Very few materials are required for vibratome sectioning, and it is a relatively straightforward procedure that is quickly mastered. This protocol describes basic vibratome sectioning that can be used for any staining procedure typically used in histology., (© 2017 Cold Spring Harbor Laboratory Press.)

Embryonic cortical neural stem cells are self-renewing progenitors that can differentiate into neurons and glia. We generated neurospheres from the developing cerebral cortex using a mouse genetic model that allows for lineage selection and found that the self-renewing neural stem cells are restricted to Sox2 expressing cells. Under normal conditions, embryonic cortical neurospheres are heterogeneous with regard to Sox2 expression and contain astrocytes, neural stem cells, and neural progenitor cells sufficiently plastic to give rise to neural crest cells when transplanted into the hindbrain of E1.5 chick and E8 mouse embryos. However, when neurospheres are maintained under lineage selection, such that all cells express Sox2, neural stem cells maintain their Pax6(+) cortical radial glia identity and exhibit a more restricted fate in vitro and after transplantation. These data demonstrate that Sox2 preserves the cortical identity and regulates the plasticity of self-renewing Pax6(+) radial glia cells.

Proper craniofacial development begins during gastrulation and requires the coordinated integration of each germ layer tissue (ectoderm, mesoderm, and endoderm) and its derivatives in concert with the precise regulation of cell proliferation, migration, and differentiation. Neural crest cells, which are derived from ectoderm, are a migratory progenitor cell population that generates most of the cartilage, bone, and connective tissue of the head and face. Neural crest cell development is regulated by a combination of intrinsic cell autonomous signals acquired during their formation, balanced with extrinsic signals from tissues with which the neural crest cells interact during their migration and differentiation. Although craniofacial anomalies are typically attributed to defects in neural crest cell development, the cause may be intrinsic or extrinsic. Therefore, we performed a phenotype-driven ENU mutagenesis screen in mice with the aim of identifying novel alleles in an unbiased manner, that are critically required for early craniofacial development. Here we describe 10 new mutant lines, which exhibit phenotypes affecting frontonasal and pharyngeal arch patterning, neural and vascular development as well as sensory organ morphogenesis. Interestingly, our data imply that neural crest cells and endothelial cells may employ similar developmental programs and be interdependent during early embryogenesis, which collectively is critical for normal craniofacial morphogenesis. Furthermore our novel mutants that model human conditions such as exencephaly, craniorachischisis, DiGeorge, and Velocardiofacial sydnromes could be very useful in furthering our understanding of the complexities of specific human diseases., (Copyright © 2011 Wiley-Liss, Inc.)

The SLP-76 family of immune cell-specific adaptors is composed of three distinct members named SLP-76, Blnk, and Clnk. They have been implicated in the signaling pathways coupled to immunoreceptors such as the antigen receptors and Fc receptors. Previous studies using gene-targeted mice and deficient cell lines showed that SLP-76 plays a central role in T-cell development and activation. Moreover, it is essential for normal mast cell and platelet activation. In contrast, Blnk is necessary for B-cell development and activation. While the precise function of Clnk is not known, it was reported that Clnk is selectively expressed in mast cells, natural killer (NK) cells, and previously activated T-cells. Moreover, ectopic expression of Clnk was shown to rescue T-cell receptor-mediated signal transduction in an SLP-76-deficient T-cell line, suggesting that, like its relatives, Clnk is involved in the positive regulation of immunoreceptor signaling. Stimulatory effects of Clnk on immunoreceptor signaling were also reported to occur in transfected B-cell and basophil leukemia cell lines. Herein, we attempted to address the physiological role of Clnk in immune cells by the generation of Clnk-deficient mice. The results of our studies demonstrated that Clnk is dispensable for normal differentiation and function of T cells, mast cells, and NK cells. Hence, unlike its relatives, Clnk is not essential for normal immune functions.

The formation of the central nervous system is one of the most fascinating processes in biology. Motor coordination, sensory perception and memory all depend on the complex cell connections that form with extraordinary precision between distinct nerve cell types within the central nervous system. The development of the central nervous system and its intricate connections occurs in several steps. During the first step known as neural induction, the neural plate forms as a uniform sheet of neuronal progenitors. Neural induction is followed by neurulation, the process in which the two halves of the neural plate are transformed into a hollow tube. Neurulation is accompanied by regionalisation of the neural tube anterior-posteriorly into the brain and spinal cord and dorso-ventrally into neural crest cells and numerous classes of sensory and motor neurons. The proper development of the vertebrate central nervous system requires the precise, finely balanced control of cell specification and proliferation, which is achieved through the complex interplay of multiple signaling systems. Bone morphogenetic proteins (BMPs), retinoic acid (RA) fibroblast growth factors (FGFs), Wnt and Hedgehog proteins are a few key factors that interact to pattern the developing central nervous system. In this review, we detail our current knowledge of the roles of these signaling factors in the development of the vertebrate nervous system in terms of the mechanisms underlying the formation and specification of the hindbrain and spinal cord.