Lanying Song

Ocular coloboma and dorsoventral neuroretinal patterning defects in Lrp6 mutant eyes.

Coloboma, an ocular birth defect seen in humans and other species, is caused by incomplete closure of the optic fissure. Here, we demonstrate that genetic deletion of Lrp6, a bottleneck coreceptor in the canonical Wnt signaling pathway, results in ocular coloboma and neuroretinal patterning defects in mice. The expression of ventral neuroretinal patterning gene Vax2 was conserved but with dorsally shifted expression domains; however, the dorsal neuroretinal patterning gene Tbx5 was lost in the Lrp6‐mutant eyes at embryonic day 10.5. Both Bmp4 and phosphorylated Smad 1/5/8 were also significantly attenuated in the dorsal neuroretina. In addition, the retinoic acid synthesizing enzymes Raldh1 and Raldh3 were significantly changed in the mutant eyes. Our findings suggest that defective retinal patterning causes coloboma in the Lrp6‐deficient mice, and that canonical Wnt signaling plays a primary role in dorsal neuroretinal patterning and related morphogenetic movements by regulation of both Bmp and retinoic acid signaling pathways. Developmental Dynamics 237:3681–3689, 2008.

The canonical Wnt/ß-catenin signaling pathway regulates Fgf signaling for early facial development

The canonical Wnt/β-catenin signaling pathway has implications in early facial development; yet, its function and signaling mechanism remain poorly understood. We report here that the frontonasal and upper jaw primordia cannot be formed after conditional ablation of β-catenin with Foxg1-Cre mice in the facial ectoderm and the adjacent telencephalic neuroepithelium. Gene expression of several cell-survival and patterning factors, including Fgf8, Fgf3, and Fgf17, is dramatically diminished in the anterior neural ridge (ANR, a rostral signaling center) and/or the adjacent frontonasal ectoderm of the β-catenin conditional mutant mice. In addition, Shh expression is diminished in the ventral telencephalon of the mutants, while Tcfap2a expression is less affected in the facial primordia. Apoptosis occurs robustly in the rostral head tissues following inactivation of Fgf signaling in the conditional mutants. Consequently, the upper jaw, nasal, ocular and telencephalic structures are absent, but the tongue and mandible are relatively developed in the conditional mutants at birth. Using molecular biological approaches, we demonstrate that the Fgf8 gene is transcriptionally targeted by Wnt/β-catenin signaling during early facial and forebrain development. Furthermore, we show that conditional gain-of-function of β-catenin signaling causes drastic upregulation of Fgf8 mRNA in the ANR and the entire facial ectoderm, which also arrests facial and forebrain development. Taken together, our results suggest that canonical Wnt/β-catenin signaling is required for early development of the mammalian face and related head structures, which mainly or partly acts through the initiation and modulation of balanced Fgf signaling activity.

Generation of Lrp6 conditional gene-targeting mouse line for modeling and dissecting multiple birth defects/congenital anomalies

Lrp6 is a key coreceptor in the canonical Wnt pathway that is widely involved in tissue/organ morphogenesis. We generated a loxP‐floxed Lrp6 mouse line. Crossing with a general Cre deleter, we obtained the Lrp6‐floxdel mice, in which the loxP‐floxed exon 2 of Lrp6 gene has been deleted ubiquitously. The homozygotes of Lrp6‐floxdel mice reproduced typical defects as seen in the conventional Lrp6‐deficient mice, such as defects in eye, limb, and neural tube, and die around birth. We also found new phenotypes including cleft palate and agenesis of external genitalia in the Lrp6‐floxdel mice. In addition, the Lrp6‐deficient embryos are known to be defective in other systems and internal organs including the heart and brain. Thus, by selectively crossing with a lineage‐specific or inducible Cre mouse line, the Lrp6 conditional gene‐targeting mice will allow us to model specific types of birth defects for mechanism and prevention studies. Developmental Dynamics 239:318–326, 2010.

Cardiac neural crest and outflow tract defects in Lrp6 mutant mice.

The role of a key Wnt coreceptor Lrp6 during heart development remains unclear. Here we show that ablation of Lrp6 in mice causes conotruncal anomalies including double‐outlet right ventricle (DORV), outflow tract (OFT) cushion hypoplasia, and ventricular septal defect (VSD). Cardiac neural crest cells are specifically lost in the dorsal neural tube and caudal pharyngeal arches of the mutant embryos. We also demonstrate that Lrp6 is required for proliferation and survival of cardiac progenitors and for the expression of Isl1 in the secondary heart field. Other known cardiogenic regulators such as Msx1, Msx2, and Fgf8 are also significantly diminished in the mutant pharyngeal arches and/or OFT. Unexpectedly, the myocardium differentiation factors Mef2c and Myocardin are upregulated in the mutant OFT. Our results indicate that Lrp6 is essential for cardiac neural crest and OFT development upstream of multiple important cardiogenic genes in different cardiac lineage cells during early cardiogenesis. Developmental Dynamics 239:200–210, 2010.

Activation of the Wnt/β-catenin Signaling Reporter in Developing Mouse Olfactory Nerve Layer Marks A Specialized Subgroup of Olfactory Ensheathing Cells

Wnt reporter TOPgal mice carry a β‐galactosidase (βgal) gene under the control of the Wnt/β‐catenin signaling responsive elements. We found that the intensely immunolabeled βgal+ cells were co‐immunolabeled with Nestin and formed a tangentially oriented single‐cell layer in the “connecting or docking zone” where the olfactory sensory axons attached to the brain surface during mid‐gestation. During early postnatal development, βgal+ cells were located in the inner olfactory nerve layer (ONLi) and co‐labeled with olfactory ensheathing cell (OEC) markers S100β and NPY but not with lineage‐specific markers for neurons, oligodendrocytes, astrocytes, and microglia, demonstrating that the TOPgal marked a subpopulation of OECs. By confocal microscopy, we found that TOPgal activated processes extended along the developing glomerulus and formed multiple tunnel‐like structures that ensheathe and bridge olfactory sensory axonal bundles from ONLi to the glomerulus, which may play a key role in glomerulus formation and convergent sorting of the peripheral olfactory axons. Developmental Dynamics 237:3157–3168, 2008.

Mutant Twinkle increases dopaminergic neurodegeneration, mtDNA deletions and modulates Parkin expression

Parkinsons disease (PD) is the second most common neurodegenerative disorder in the developed world, and is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Somatic mitochondrial DNA (mtDNA) deletions reach their highest concentration with age in the SN in humans, and may contribute to PD; yet whether mtDNA deletions cause DA neuron degeneration remains unclear. Inherited mutations of Twinkle helicase involved in mtDNA replication causes a dominant increase in mtDNA deletions in humans. We constructed a mouse model expressing mutant Twinkle in DA neurons. Mutant mice had an increase in age-related mtDNA deletions, reduction of DA neuron number in SN at 17-22 months and displayed abnormalities in rota-rod behavior. Functional analysis of midbrain indicated a slight reduction in mitochondrial state II respiration in mutants, but no decrease in maximal respiration. Also, Parkin expression was significantly decreased in DA neurons in the SN of 22-month-old mutant mice, and in PC12 cells after 48 h transfection of mutant Twinkle. Both confocal imaging and coimmunoprecipitation indicated interaction of Twinkle with Parkin in the mitochondria. Parkin overexpression rescued the reduction of proteasome activity caused by mutant Twinkle in PC12 cells. In addition, the autophagy marker LC3 was increased in the SN of 22-month transgenics, and this increase was similarly mutant Twinkle-dependent in PC12 cells. Collectively, our data demonstrate that mammalian Twinkle is important for mitochondrial integrity in DA neurons and provide a novel mouse model in which increased mtDNA deletions may lead to DA neuron degeneration and parkinsonism.

Mitochondrial complex I defects increase ubiquitin in substantia nigra

Parkinson׳s disease (PD) is the second most common neurodegenerative disorder in the developed world, and is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN) of midbrain. Mitochondrial complex I dysfunction has been implicated in PD pathophysiology, yet the molecular mechanism by which complex I defects may cause DA neurodegeneration remain unclear. Using Ndufs4 mouse model of mitochondrial complex I deficiency, we observed a remarkable ubiquitin protein increase in SN of Ndufs4-/- (KO) mice. By contrast, neurofilaments were significantly decreased in SN of KO mice. Furthermore, mass spectrometry and co-immunoprecipitation (Co-IP) analysis indicated an increase in ubiquitinated neurofilaments in midbrain of KO mice, whereas 20S proteasome activities were decreased, which could potentially explain the buildup of ubiquitin protein. Collectively, these data suggest that mitochondrial complex I defects cause proteasome inhibition, a consequent increase in ubiquitinated neurofilaments and other proteins, and decrease the expression of neurofilaments that could be relevant to the mechanism of DA neuronal death in PD.