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Cerebellum is one of the major targets of autoimmunity and cerebellar damage that leads to ataxia characterized by the loss of fine motor coordination and balance, with no treatment available. Deep brain stimulation (DBS) could be a promising treatment for ataxia but has not been extensively investigated. Here, our study aims to investigate the use of interposed nucleus of deep cerebellar nuclei (IN-DCN) for ataxia. We first characterized ataxia-related motor symptom of a Purkinje cell (PC)-specific LIM homeobox (Lhx)1 and Lhx5 conditional double knockout mice by motor coordination tests, and spontaneous electromyogram (EMG) recording. To validate IN-DCN as a target for DBS, in vivo local field potential (LFP) multielectrode array recording of IN-DCN revealed abnormal LFP amplitude surges in PCs. By synchronizing the EMG and IN-DCN recordings (neurospike and LFP) with high-speed video recordings, ataxia mice showed poorly coordinated movements associated with low EMG amplitude and aberrant IN-DCN neural firing. To optimize IN-DCN-DBS for ataxia, we tested DBS parameters from low (30 Hz) to high stimulation frequency (130 or 150 Hz), and systematically varied pulse width values (60 or 80 µs) to maximize motor symptom control in ataxia mice. The optimal IN-DCN-DBS parameter reversed motor deficits in ataxia mice as detected by animal behavioral tests and EMG recording. Mechanistically, cytokine array analysis revealed that anti-inflammatory cytokines such as interleukin (IL)-13 and IL-4 were upregulated after IN-DCN-DBS, which play key roles in neural excitability. As such, we show that IN-DCN-DBS is a promising treatment for ataxia and possibly other movement disorders alike.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by social communication deficit and repetitive behaviour. In the past few years, increasing clinical evidence has shown that the cerebellum may contribute to the neuropathology of ASD. However, studies in the mechanism for the involvement of the cerebellum in autism remained speculative. Although some have suggested the possibility of a change of glutamate decarboxylases in the cerebellum of autistic patients, this remains controversial and is limited to the alteration in transcriptional level. This study aimed to investigate the cerebellar structure and determine the expression of rate-limiting GABAergic enzymes in GABA signalling of the autism cerebellum. Pregnant C57BL/6 J mice were intraperitoneally injected with a dosage of 500 mg/kg valproic acid (VPA) on embryonic day 10.5 for autistic behavioural induction. This study found that early prenatal exposure to VPA led to tail deformation and decreased cerebellar weight and size. Early adult mouse models with autistic behaviour showed reduced expression of both isoforms of glutamate decarboxylases (GAD) 65 and 67 in the cerebellum. Also, protein expressions of cerebellar type 1 GABA transporter (GAT-1) and GABA transaminase (GABAT) were reduced in VPA mice. It indicated that abnormal GABA production, recycling, and metabolism could alter the excitatory-inhibitory balance in the autistic cerebellum. Thus, our findings provide supporting evidence that cerebellum impairment could be an etiology of environmentally induced autism. Changes in cerebellar structure and the altered GABAergic enzymes in the cerebellum provide targets for future therapeutic studies in idiopathic autism.

Polyglutamine diseases are a set of progressive neurodegenerative disorders caused by misfolding and aggregation of mutant CAG RNA and polyglutamin protein. To date, there is a lack of effective therapeutics that can counteract the polyglutamine neurotoxicity. Two peptidylic inhibitors, QBP1 and P3, targeting the protein and RNA toxicities, respectively, have been previously demonstrated by us with combinational therapeutic effects on the Drosophila polyglutamine disease model. However, their therapeutic efficacy has never been investigated in vivo in mammals. The current study aims to (a) develop a brain-targeting delivery system for both QBP1 and L1P3V8 (a lipidated variant of P3 with improved stability) and (b) evaluate their therapeutic effects on the R6/2 transgenic mouse model of polyglutamine disease. Compared with intravenous administration, intranasal administration of QBP1 significantly increased its brain-to-plasma ratio. In addition, employment of a chitosan-containing in situ gel for the intranasal administration of QBP1 notably improved its brain concentration for up to 10-fold. Further study on intranasal cotreatment with the optimized formulation of QBP1 and L1P3V8 in mice found no interference on the brain uptake of each other. Subsequent efficacy evaluation of 4-week daily QBP1 (16 μmol/kg) and L1P3V8 (6 μmol/kg) intranasal cotreatment in the R6/2 mice demonstrated a significant improvement on the motor coordination and explorative behavior of the disease mice, together with a full suppression on the RNA- and protein-toxicity markers in their brains. In summary, the current study developed an efficient intranasal cotreatment of the two peptidylic inhibitors, QBP1 and L1P3V8, for their brain-targeting, and such a novel therapeutic strategy was found to be effective on a transgenic polyglutamine disease mouse model.

The transcription factor Brachyury (T) gene is expressed throughout primary mesoderm (primitive streak and notochord) during early embryonic development and has been strongly implicated in the genesis of chordoma, a sarcoma of notochord cell origin. Additionally, T expression has been found in and proposed to play a role in promoting epithelial-mesenchymal transition (EMT) in various other types of human tumors. However, the role of T in normal mammalian notochord development and function is still not well-understood. We have generated an inducible knockdown model to efficiently and selectively deplete T from notochord in mouse embryos. In combination with genetic lineage tracing, we show that T function is essential for maintaining notochord cell fate and function. Progenitors adopt predominantly a neural fate in the absence of T, consistent with an origin from a common chordoneural progenitor. However, T function is dispensable for progenitor cell survival, proliferation, and EMT, which has implications for the therapeutic targeting of T in chordoma and other cancers.

Background: Shellfish hypersensitivity is among the most common food allergies. A murine model of IgE-mediated shrimp allergy has been established in our laboratory. The aim of this study is to determine the intestinal histological changes and cytokine expression profile of this model sensitized with the major shellfish allergen tropomyosin. Methods: Female Balb/c mice orally sensitized and challenged with recombinant tropomyosin were sacrificed. Continuous sections of duodenum, jejunum and ileum were prepared using the Swiss roll technique for histological and immunological analysis. Duodenal epithelial cell apoptosis and migration were examined. mRNA expression of IL-4, IL-6, IL-10, IL-13, IL-18 and IFN-γ in intestinal tissue was measured via RT-PCR. Results: In tropomyosin-sensitized and challenged mice, an increased number of eosinophils, mast cells and goblet cells was found 24 h after challenge. There were also increased mast cell and goblet cell numbers at 72 h after challenge, but the level of eosinophils decreased. Differences compared with control mice are most prominent at the duodenum compared to the distal regions. In addition, TUNEL assay indicates a significantly higher apoptosis rate in sensitized mice sacrificed 72 h after challenge, and mRNA expression showed a biased Th2/Th1 cytokine profile and a higher level of murine mast cell protease 1. Conclusions: This study documented a multitude of histological and immunological changes in the gut in a murine model of shrimp allergy. Even without repetitive intragastric challenge, shrimp tropomyosin induces an increase in the number of inflammatory cells to varying degrees within the small intestine. This model provides an important tool for testing new therapeutic interventions.

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

The expression of bone morphogenic protein 4 (BMP4), a new pro-inflammatory marker, is increased by disturbed flow in endothelial cells (ECs). BMP4 stimulates production of reactive oxygen species (ROS) and causes endothelial cell dysfunction. The present study examined BMP4-induced apoptosis in ECs and isolated arteries from rat, mouse, and human, and the signaling pathways mediating BMP4-induced apoptosis. Apoptosis was assessed by flow cytometry to detect Annexin-V positive cells, and terminal deoxynucleotidyl transferase dUTP nick end (TUNEL) labeling. The superoxide production was measured by dihydroethidium fluorescence. BMP4 induced EC apoptosis in human mesenteric arteries, mouse aortic endothelium, rat primary ECs, and human ECs. BMP4-induced EC apoptosis was mediated through ROS production by activation of NADPH oxidase, which led to cleaved caspase-3 expression. BMP4 also induced sequential activation of p38 MAPK and JNK which was upstream of caspase 3 activation. Knockdown of BMP receptor 1A by lentiviral shRNA or NOX4 siRNA transfection inhibited BMP4-induced ROS production, p38 and JNK phosphorylation, and caspase-3 activation in ECs. JNK siRNA inhibited BMP4-induced JNK phosphorylation and caspase-3 activation. The present study delineates that BMP4 causes EC apoptosis through activation of caspase-3 in a ROS/p38MAPK/JNK-dependent signaling cascade.

Retinal neurons extend their dendritic fields to achieve a degree of dendritic overlap with homotypic neighbors that is cell-type specific. How these neurons regulate their dendritic growth is unclear. The dendritic field of a retinal horizontal cell varies inversely with horizontal cell density across different strains of mice, suggesting that proximity to neighboring cells regulates dendritic growth. To test this directly, we have employed the Cre-loxP conditional gene targeting strategy to achieve inactivation of Lim1 function in developing horizontal cells. Through this approach, Lim1 function was prevented within a subset of horizontal cells that in turn fail to migrate to the horizontal cell layer and differentiate normally. For those remaining horizontal cells with Lim1 intact (about half of the normal population in these mice), we show that they spread themselves out tangentially and differentiate a dendritic morphology that is essentially normal but for the fact that it has nearly doubled in area. Such larger horizontal cells, sampling from an area of retina containing twice their normal afferent number, differentiate a dendritic field with nearly double the number of higher order branches and terminal clusters. These results demonstrate directly that positioning and dendritic growth are regulated by interactions with homotypic neighbors, whereas afferents instruct the differentiation of dendritic patterning.

Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5 , were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1 , which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum.

Summary: Hair differentiation and growth are controlled by complex reciprocal signaling between epithelial and mesenchymal cells. To better understand the requirement and molecular mechanism of BMP signaling in hair follicle development, we performed genetic analyses of bone morphogenetic protein receptor 1A (BMPR-IA) function during hair follicle development by using a conditional knockout approach. The conditional mutation of Bmpr1a in ventral limb ectoderm and its derivatives (epidermis and hair follicles) resulted in a lack of hair outgrowth from the affected skin regions. Mutant hair follicles exhibited abnormal morphology and lacked hair formation and pigment deposition during anagen. The timing of the hair cycle and the proliferation of hair matrix cells were also affected in the mutant follicles. We demonstrate that signaling via epithelial BMPR-IA is required for differentiation of both hair shaft and inner root sheath from hair matrix precursor cells in anagen hair follicles but is dispensable for embryonic hair follicle induction. Surprisingly, aberrant de novo hair follicle morphogenesis together with hair matrix cell hyperplasia was observed in the absence of BMPR-IA signaling within the affected skin of adult mutants. They developed hair follicle tumors from 3 months of age, indicating that inactivation of epidermal BMPR-IA signaling can lead to hair tumor formation. Taken together, our data provide genetic evidence that BMPR-IA signaling plays critical and multiple roles in controlling cell fate decisions or maintenance, proliferation, and differentiation during hair morphogenesis and growth, and implicate Bmpr1a as a tumor suppressor in skin tumorigenesis. genesis 39:10–25, 2004. © 2004 Wiley-Liss, Inc.

Recent advances using site-specific recombinase systems to manipulate the mouse genome have opened new doors, revealing the complexity of gene function in mammals and for designing better models of human diseases (Torres and Kuhn, 1997; Dymecki, 2000). The application of these site-specific recombinase systems along with gene targeting techniques in embryonic stem (ES) cells have now made it possible to modify the mouse genome in almost any desired manner, from creating specific point mutations to achieving large sitespecific chromosomal rearrangements (Nagy and Mar, 2001; Yu and Bradley, 2001). Furthermore, these techniques have proven to be very useful tools for activating and inactivating specific genes in a “conditional” manner in spatially and temporally restricted patterns (Lobe and Nagy, 1998; Nagy, 2000). This helps to circumvent the limitations of conventional gene inactivation, in particular, that a given gene has multiple roles at different times and in different tissues. These methodologies are particularly useful for elucidating a complete picture of gene function in which: 1) the conventional knockout leads to an early lethal phenotype preventing the study of its later roles, and/or 2) the phenotype affects multiple tissues, preventing the detailed study of its function in a particular cell lineage.

The lysosome is a membrane-bound organelle involved in the turnover of various intracellular and extracellular macromolecules. These are degraded by acidic hydrolases in the lumen of lysosome. The lysosomal membrane is important not only in retaining the acidic hydrolases to protect cells against cytosolic proteolysis, but it also facilitates protein trafficking though organelle fusion. In this study, we report on a novel lysosomal membrane protein transmembrane 6 superfamily 1 (Tm6sf1). Expression of Tm6sf1-DsRed fusion proteins in HEK293A cells displayed punctate or ringlike vesicles, which colocalized with conventional lysosome markers including LAMP1/2, RAB7, and Rnf167. Using fluorescence time-lapse live cell imaging, we demonstrated the fusion of Tm6sf1 vesicles with lysosomes and the integration of Tm6sf1 into the lysosomal membrane. We also examined the expression of Tm6sf1 in mouse tissues and found immunopositive signals in major organs such as the cerebellum, kidney, and intestine. These data suggest that Tm6sf1 is a widely expressed lysosomal transmembrane protein and can be used as a novel marker of lysosome.

There are conflicting views on whether collagen X is a purely structural molecule, or regulates bone mineralization during endochondral ossification. Mutations is the human collagen α1(X) gene (COL10A1) in Schmid metaphyseal chondrodysplasia (SMCD) suggest a supportive role. But mouse collagen α1(X) gene (Col10A1) null mutants were previously reported to show no obvious phenotypic change. We have generated collagen X deficient mice, which shows that deficiency does have phenotypic consequences which partly resemble SMCD, such as abnormal trabecular bone architecture. In particular, the mutant mice develop coxa vara, a phenotypic change common in human SMCD. Other consequences of the mutation are reduction in thickness of growth plate resting zone and articular cartilage, altered bone content, and atypical distribution of matrix components within growth plate cartilage. We propose that collagen X plays a role in the normal distribution of matrix vesicles and proteoglycans within the growth plate matrix. Collagen X deficiency impacts on the supporting properties of the growth plate and the mineralization process, resulting in abnormal trabecular bone. This hypothesis would accommodate the previously conflicting views of the function of collagen X and of the molecular pathogenesis of SMCD., published_or_final_version

Objective— Bone morphogenic protein 4 (BMP4) is involved in the development of endothelial dysfunction in hypertension. This study investigated whether the inhibition of BMP4 signaling improves endothelial function in db/db diabetic mice. Approach and Results— Male db/db mice were treated with noggin via osmotic pump infusion (1 µg/[h·kg –1 ]) for 2 weeks. Adenovirus BMP4-short hairpin RNA was introduced via tail vein injection at a dosage of 10 9 pfu/mouse and its effects were examined 7 days after. Vasoreactivity was studied on wire and pressure myograph. Both noggin treatment and adenovirus BMP4-short hairpin RNA transduction improved endothelium-dependent relaxations in aortae and flow-mediated dilatation in mesenteric arteries of db/db mice. Ex vivo treatment with BMP4 inhibitors and adenovirus BMP4-short hairpin RNA rescued the impaired endothelium-dependent relaxations in db/db mouse aortae and reduced reactive oxygen species overproduction determined by dihydroethidium staining, CM-H 2 DCFDA fluorescence imaging, and chemiluminescence assay in db/db mouse aortae, and also in ex vivo cultured C57BL/6 mouse aortae or primary mouse aortic endothelial cells treated with high glucose. Likewise, activin receptor–like kinase 3 silencing by short hairpin RNA lentivirus improved endothelium-dependent relaxations in db/db mouse aortae accompanied by reactive oxygen species inhibition in endothelial cells. In addition, noggin reduced BMP4 upregulation in high-glucose–treated endothelial cells and in C57BL/6 mouse aortae and in aortae from db/db mice. Conclusions— Inhibition of BMP4/activin receptor–like kinase 3/reactive oxygen species signaling improved endothelial function in diabetic mice through limiting oxidative stress in endothelium. Inhibiting BMP4 cascade can become another potential therapeutic strategy against diabetic vascular dysfunction.

Bone morphogenetic protein (BMP) signaling is critical for cerebellum development. However, the details of receptor regulated-Smad (R-Smad) and common partner Smad (Co-Smad, or Smad4) involvement are unclear. Here, we report that cerebellum-specific double conditional inactivation of Smad1 and Smad5 (Smad1/5) results in cerebellar hypoplasia, reduced granule cell numbers, and disorganized Purkinje neuron migration during embryonic development. However, single conditional inactivation of either Smad1 or Smad5 did not result in cerebellar abnormalities. Surprisingly, conditional inactivation of Smad4, which is considered to be the central mediator of canonical BMP-Smad signaling, resulted only in very mild cerebellar defects. Conditional inactivation of Smad1/5 led to developmental defects in the anterior rhombic lip (ARL), as shown by reduced cell proliferation and loss of Pax6 and Atoh1 expression. These defects subsequently caused the loss of the nuclear transitory zone and a region of the deep cerebellar nuclei. The normal maturation of the remaining granule cell precursors in the external granular layer (EGL) suggests Smad1/5 signaling is required for the specification process in ARL but not for the subsequent EGL development. Our results demonstrate functional redundancy for Smad1 and Smad5 but functional discrepancy between Smad1/5 and Smad4 during cerebellum development.

LIM1 (also known as LHX1) is a LIM-class homeodomain protein that has been shown by gene targeting to play essential roles in the formation of head, kidney, and gonads during mouse embryogenesis (Shawlot and Behringer, 1995). In early mouse development, Lim1 is expressed in the extraembryonic anterior visceral endoderm (AVE) and in the embryonic primitive streak and the newly formed mesoderm during gastrulation (Shawlot and Behringer, 1995; Tsang et al., 2000). Lim1 activity is required in both primitive streak-derived tissues and visceral endoderm for proper head formation by regulating sequential signaling events (Shawlot et al., 1999). However, Lim1 is also expressed widely after gastrulation in the developing central nervous system from telencephalon, diencephalon, hindbrain through spinal cord, and in several urogenital tissues such as the mesonephros, mesonephric (Wolffian) ducts, uteric buds, and definitive kidney (Barnes et al., 1994; Fujii et al., 1994; Karavanov et al., 1996). These observations implicate the Lim1 gene in the specification and/or maintenance of multiple cell lineages. Recent studies using chimera technology indeed show that Lim1 is important in the selection of axonal projections of lateral motor column (LMC) neurons in the developing limb (Kania et al., 2000). However most Lim1-deficient mice die at midgestation, around embryonic day 10 (E10) with a headless phenotype. Its functions later in development remains unclear. To dissect the functions of Lim1 in these different tissues and organs, we employed the Cre-loxP conditional gene targeting strategy to inactivate Lim1 function in a tissue-specific manner (Nagy, 2000). A conditional allele of Lim1 with two loxP sites flanking the entire coding region (Lim1-floxed allele) was made by gene targeting in mouse embryonic stem (ES) cells followed by subsequent crosses with a CMV-Cre transgenic mouse line (Cheah et al., 2000) to remove the neo selection cassette (Figs. 1, 2A). To demonstrate that the Lim1floxed allele can recombine correctly in vivo, we have crossed mice heterozygous for the Lim1-floxed allele with a Mox2-Cre mouse line which expresses Cre recombinase throughout the epiblast following implantation during development (Tallquist and Soriano, 2000). Correct recombination of the Lim1-floxed allele producing the Lim1-deleted allele (leaving one loxP site) was detected using PCR genotyping (Fig. 2B). Mice homozygous for the Lim1-deleted allele phenocopy the headless phenotype of Lim1 null mice (Fig. 2C) (Shawlot and Behringer, 1995). Thus, we demonstrate that Cre-mediated recombination of the Lim1-floxed allele in vivo results in a Lim1 null allele.

Mouse embryos lacking Lhx1 (Lim1) activity display defective gastrulation and are deficient of primordial germ cells (PGCs) (Tsang et al. [2001] International Journal of Developmental Biology 45:549-555). To dissect the specific role of Lhx1 in germ cell development, we studied embryos with conditional inactivation of Lhx1 activity in epiblast derivatives, which, in contrast to completely null embryos, develop normally through gastrulation before manifesting a head truncation phenotype. Initially, PGCs are localized properly to the definitive endoderm of the posterior gut in the conditional mutant embryos, but they depart from the embryonic gut prematurely. The early exit of PGCs from the gut is accompanied by the failure to maintain a strong expression of Ifitm1 in the mesoderm enveloping the gut, which may mediate the repulsive activity that facilitates the retention of PGCs in the hindgut during early organogenesis. Lhx1 therefore may influence the localization of PGCs by modulating Ifitm1-mediated repulsive activity.

Purkinje cells (PCs) are one of the principal neurons in the cerebellar cortex that play a central role in the coordination of fine-tuning body movement and balance. To acquire normal cerebellum function, PCs develop extensive dendritic arbors that establish synaptic connections with the parallel fibers of granule cells to form the proper neuronal circuitry. Therefore, dendritic arborization of PCs is an important developmental step to construct the mature neural network in the cerebellum. However, the genetic control of this process is not fully understood. In this study, Foxp4, a forkhead transcription factor that is expressed specifically in migrating and mature PCs of cerebellum from embryonic stages to adulthood, was knocked down by small interfering RNA (siRNA) in organotypic cerebellar slice culture. When Foxp4 expression was knocked down at postnatal day 5 (P5), no abnormalities for early dendritic remodeling of PCs were observed. However, when Foxp4 was knocked down in P10 cerebellar slices, the organization of PC dendritic arbors was highly impaired, leaving hypoplastic but non-apoptotic cell bodies. The radial alignment of Bergmann glial fibers that associated with PC dendrites was also lost. These results suggest that Foxp4 is dispensable for the early PC dendrite outgrowth, but is essential for the maintenance of PC dendritic arborization and subsequent association with Bergmann glial fibers.

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Mullerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Mullerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Mullerian hormone, which activates the regression of the Mullerian duct. Anti-Mullerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Mullerian hormone receptor 2 (AMHR2), in the Mullerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Mullerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Mullerian hormone signal required for Mullerian duct regression. Loss of these genes in the Mullerian duct mesenchyme results in male infertility due to retention of Mullerian duct derivatives in an otherwise virilized male.

Although much is known about the transcriptional regulation that coordinates retinal cell fate determination, very little is known about the developmental processes that establish the characteristic laminar architecture of the retina, in particular, the specification of neuronal positioning. The LIM class homeodomain transcription factor Lim1 (Lhx1) is expressed in postmitotic, differentiating, and mature retinal horizontal cells. We show that conditional ablation ofLim1results in the ectopic localization of horizontal cells to inner aspects of the inner nuclear layer, among the retinal amacrine cells. The ectopic cells maintain a molecular phenotype consistent with horizontal cell identity; however, these neurons adopt a unique morphology more reminiscent of amacrine cells, including a dendritic arbor positioned within the inner plexiform layer. All other retinal cell populations appear unaltered. Our data suggest a model wherebyLim1lies downstream of horizontal cell fate determination factors and functions cell autonomously to instruct differentiating horizontal cells to the appropriate laminar position in the developing retina. This study is the first to describe a cell type-specific genetic program that is essential for targeting a discrete retinal neuron population to the proper lamina.

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded α1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia., Author Summary The assembly and folding of secreted proteins in the endoplasmic reticulum (ER) is exquisitely regulated by a complex mechanism that maintains an equilibrium between folded and unfolded proteins. Perturbation of this homeostasis induces ER stress, which, if not alleviated through ER stress signaling (ERSS), ultimately triggers cell death. Normal bone growth occurs through a highly coordinated differentiation program that yields specialized cartilage cells (chondrocytes); when this program is disrupted, chondrodysplasia, or malformed skeletons, can result. Chondrodysplasias caused by mutations that affect protein assembly and secretion are characterized by a disorganization of bony growth plates and distension of the ER. We tested whether these chondrodysplasia characteristics were linked to ERSS. By investigating the impact of ER stress on the cell fate of hypertrophic chondrocytes (HCs) in transgenic mice expressing mutations in collagen that prevent proper folding, we revealed a novel adaptive mechanism that helps alleviate the unfolded protein load. Instead of undergoing apoptosis, the HCs undergoing ER stress adapt, re-enter the cell cycle, and revert to a less-mature state in which expression of the mutant collagen is reduced. Our findings have broad implications for adaptive mechanisms to ER stress in vivo and for the pathophysiology underlying chondrodysplasias caused by mutations that impact on protein assembly and secretion., When subjected to ER stress (by expression of misfolded or unfolded proteins), hypertrophic chondrocytes undergo alterations to their developmental program that may be part of an adaptive response.

Kidney organogenesis requires the morphogenesis of epithelial tubules. Inductive interactions between the branching ureteric buds and the metanephric mesenchyme lead to mesenchyme-to-epithelium transitions and tubular morphogenesis to form nephrons, the functional units of the kidney. The LIM-class homeobox gene Lim1 is expressed in the intermediate mesoderm, nephric duct, mesonephric tubules, ureteric bud, pretubular aggregates and their derivatives. Lim1-null mice lack kidneys because of a failure of nephric duct formation, precluding studies of the role of Lim1 at later stages of kidney development. Here, we show that Lim1 functions in distinct tissue compartments of the developing metanephros for both proper development of the ureteric buds and the patterning of renal vesicles for nephron formation. These observations suggest that Lim1 has essential roles in multiple steps of epithelial tubular morphogenesis during kidney organogenesis. We also demonstrate that the nephric duct is essential for the elongation and maintenance of the adjacent Müllerian duct, the anlage of the female reproductive tract.

During gastrulation and early organogenesis, Lim1 is expressed in the visceral endoderm, the anterior mesendoderm, and the lateral mesoderm that comprises the lateral plate and intermediate mesoderm. A previous study has reported that kidneys and gonads are missing in the Lim1 null mutants (W. Shawlot and R. R. Behringer, 1995, Nature 374, 425–430). Results of the present study show that in the early organogenesis stage mutant embryo, the intermediate mesoderm that contains the urogenital precursor tissues is disorganized and displays diminished expression of PAX2 and the Hoxb6-lacZ transgene. When posterior epiblast cells of the Lim1 null mutant embryo were transplanted to the primitive streak of wild-type host embryos, they were able to colonize the lateral plate and intermediate mesoderm of the host, suggesting that Lim1 activity is not essential for the allocation of epiblast cells to these mesodermal lineages. However, most of the mutant cells that colonized the lateral and intermediate mesoderm of the host embryo did not express the Hoxb6-lacZ transgene, except for some cells that were derived from the distal part of the posterior epiblast. Lim1 activity may therefore be required for the full expression of this transgene that normally marks the differentiation of the lateral plate and intermediate mesoderm.

Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral endoderm and in primitive streak-derived tissues of early mouse embryos. Mice homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain. To determine in which tissues Lim1 is required for head formation and its mode of action, we have generated chimeric mouse embryos and performed tissue layer recombination explant assays. In chimeric embryos in which the visceral endoderm was composed of predominantly wild-type cells, we found that Lim1−/− cells were able to contribute to the anterior mesendoderm of embryonic day 7.5 chimeric embryos but that embryonic day 9.5 chimeric embryos displayed a range of head defects. In addition, early somite stage chimeras generated by injecting Lim1−/− embryonic stem cells into wild-type tetraploid blastocysts lacked forebrain and midbrain neural tissue. Furthermore, in explant recombination assays, anterior mesendoderm from Lim1−/− embryos was unable to maintain the expression of the anterior neural marker gene Otx2 in wild-type ectoderm. In complementary experiments, embryonic day 9.5 chimeric embryos in which the visceral endoderm was composed of predominantly Lim1−/− cells and the embryo proper of largely wild-type cells, also phenocopied the Lim1−/− headless phenotype. These results indicate that Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation and that its inactivation in these tissues produces cell non-autonomous defects. We discuss a double assurance model in which Lim1 regulates sequential signaling events required for head formation in the mouse.

The entire mouse collagen X gene (Col10a-1) has been isolated. The gene is composed of three exons and two introns spanning 7.0 kb of the DNA sequence. Exons 2 and 3 together encode 15-bp of 5' untranslated sequence, a 2040-bp open reading frame and an 895-nucleotide 3' non-coding region. In the 5' flanking region of the gene, two consensus TATA-box sequences were found. Identification of the first exon by ribonuclease-protection assays and the determination of the 5' end of Col10a-1 mRNA transcripts by primer-extension analyses show that the more 3' TATA box is probably predominantly used and that there are at least three transcription start sites in the exon 1 sequence 3' to this, resulting in 5' untranslated regions of 78, 77 and 55 nucleotides. By means of rapid amplification of cDNA ends by polymerase chain reaction, an additional mRNA species was detected which overlapped the other Col10a-1 transcripts, including the 3' TATA box sequence, giving a 5' untranslated sequence of approximately 235 bases. This latter transcript starts approximately 20 bp 3' to the more 5' TATA box. The data suggest alternative use of promoters and transcription starts for the Col10a-1 gene. Comparison of the combined nucleotide and deduced amino acid sequences of exons 2 and 3 with chicken, bovine and human collagen X genes, showed a high degree of similarity indicating conservation of this gene throughout evolution. Mouse Col10a-1 mRNA was shown to be approximately 3.0 kb and the pepsinized protein, as detected by SDS/PAGE, was approximately 45 kDa. The mRNA and protein sizes correlate with that predicted by the open reading frame. Reverse-transcription polymerase chain reaction assays indicate that the mouse collagen X gene is first expressed at 13.5 days post coitum, temporally preceding the onset of endochondral ossification. In agreement with the generally accepted association of type-X collagen with endochondral ossification, in situ hybridization analyses indicate that Col10a-1 mRNA are restricted to the hypertrophic regions of growth cartilage.

Background Lim1 is a homeobox gene that is essential for nephrogenesis. During metanephric kidney development, Lim1 is expressed in the nephric duct, ureteric buds, and the induced metanephric mesenchyme. Conditional ablation of Lim1 in the metanephric mesenchyme blocks the formation of nephrons at the nephric vesicle stage, leading to the production of small, non-functional kidneys that lack nephrons. Methods In the present study, we used Affymetrix probe arrays to screen for nephron-specific genes by comparing the expression profiles of control and Lim1 conditional mutant kidneys. Kidneys from two developmental stages, embryonic day 14.5 (E14.5) and 18.5 (E18.5), were examined. Results Comparison of E18.5 kidney expression profiles generated a list of 465 nephron-specific gene candidates that showed a more than 2-fold increase in their expression level in control kidney versus the Lim1 conditional mutant kidney. Computational analysis confirmed that this screen enriched for kidney-specific genes. Furthermore, at least twenty-eight of the top fifty (56%) candidates (or their vertebrate orthologs) were previously reported to have a nephron-specific expression pattern. Our analysis of E14.5 expression data yielded 41 candidate genes that are up-regulated in the control kidneys compared to the conditional mutants. Three of them are related to the Notch signaling pathway that is known to be important in cell fate determination and nephron patterning. Conclusion Therefore, we demonstrate that Lim1 conditional mutant kidneys serve as a novel tissue source for comprehensive expression studies and provide a means to identify nephron-specific genes.