Cerebellar granule cell migration and folia development require Mllt11/Af1q/Tcf7c.

The organization of neurons into distinct layers, known as lamination, is a common feature of the nervous system. This process, which arises from the direct coupling of neurogenesis and neuronal migration, plays a crucial role in the development of the cerebellum, a structure exhibiting a distinct folding cytoarchitecture with cells arranged in discrete layers. Disruptions to neuronal migration can lead to various neurodevelopmental disorders, highlighting the significance of understanding the molecular regulation of lamination. We report a role Mllt11/Af1q/Tcf7c (myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 fused gene from chromosome 1q, also known as Mllt11 transcriptional cofactor 7; henceforth referred to Mllt11) in the migration of cerebellar granule cells (GCs). We now show that Mllt11 plays a role in both the tangential and radial migration of GCs. Loss of Mllt11 led to an accumulation of GC precursors in the rhombic lip region and a reduction in the number of GCs successfully populating developing folia. Consequently, this results in smaller folia and an overall reduction in cerebellar size. Furthermore, analysis of the anchoring centers reveals disruptions in the perinatal folia cytoarchitecture, including alterations in the Bergmann glia fiber orientation and reduced infolding of the Purkinje cell plate. Lastly, we demonstrate that Mllt11 interacts with non-muscle myosin IIB (NMIIB) and Mllt11 loss-reduced NMIIB expression. We propose that the dysregulation of NMIIB underlies altered GC migratory behavior. Taken together, the findings reported herein demonstrate a role for Mllt11 in regulating neuronal migration within the developing cerebellum, which is necessary for its proper neuroanatomical organization.

Retinal neuroblast migration and ganglion cell layer organization require the cytoskeletal-interacting protein Mllt11.

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.

Mllt11 Regulates Migration and Neurite Outgrowth of Cortical Projection Neurons during Development.

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 gain-of-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.SIGNIFICANCE STATEMENT The regulation of cortical projection neuron (CPN) morphologies is an area of active investigation since the time of Cajal. Yet the molecular mechanisms of how the complex dendritic and axonal morphologies of projection neurons are formed remains incompletely understood. Although conditional mutagenesis analysis in the mouse, coupled with overexpression assays in the developing fetal brain, we show that a novel protein called Mllt11 is sufficient and necessary to regulate the dendritic and axonal characteristics of callosal projection neurons in the developing mammalian neocortex. Furthermore, we show that Mllt11 interacts with microtubules, likely accounting for its role in neuritogenesis.

MLLT11/AF1q is differentially expressed in maturing neurons during development.

Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11 or ALL1 fused from chromosome 1q (MLLT11/AF1q) is a highly conserved 90 amino acid protein that functions in hematopoietic differentiation. Its translocation to the Trithorax locus has been implicated in malignancies of the hematopoietic system. However, the spatio-temporal profile of MLLT11 expression during embryonic development has not been characterized. Here we show that MLLT11 has a remarkably specific expression pattern in the developing central and peripheral nervous system. We find high levels of MLLT11 transcript and protein expression in the developing marginal zone of the cortex and spinal cord. MLLT11 co-localized with Tbr2 in the developing subplate region of the cortex and expanded to encompass the cortical plate at late fetal stages. Expression in the peripheral nervous system initiated at E9.5 in the facio-acoustic cranial ganglia and elaborated to identify all the cranio-facial and dorsal root ganglia by E10.5. We also observed expression in the eye and gastrointestinal tract, where MLLT11 transcripts localized to Tuj1-positive inner retinal layer and autonomic neurons, respectively. Altogether these results show that MLLT11 is a pan-neuronal marker, suggesting a role in neural differentiation in the central nervous system and neural crest-cell derived peripheral ganglia.

Mutations in Hedgehog acyltransferase (Hhat) perturb Hedgehog signaling, resulting in severe acrania-holoprosencephaly-agnathia craniofacial defects.

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.

Flexibility of neural stem cells.

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.

A phenotype-driven ENU mutagenesis screen identifies novel alleles with functional roles in early mouse craniofacial development.

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.

Immune functions in mice lacking Clnk, an SLP-76-related adaptor expressed in a subset of immune cells.

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.

Gene expression and regulation of hindbrain and spinal cord development.

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.