Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei

<p>Abstract</p> <p>Background</p> <p>The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express <it>Sonic Hedgehog </it>(<it>Shh</it>). However, <it>Shh </it>expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene <it>Gli1 </it>initiates in the ventral midline prior to <it>Shh </it>expression, but after the onset of <it>Shh </it>expression it is expressed in precursors lateral to <it>Shh</it>-positive cells. Given these dynamic gene expression patterns, <it>Shh </it>and <it>Gli1 </it>expression could delineate different progenitor populations at distinct embryonic time points.</p> <p>Results</p> <p>We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express <it>Shh </it>(Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from <it>Shh</it>- and <it>Gli1</it>-expressing progenitors located outside of the ventral midline give rise to astrocytes.</p> <p>Conclusions</p> <p>We define a ventral midbrain precursor map based on the timing of <it>Gli1 </it>and <it>Shh </it>expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions.</p

Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.Funding for this study was provided by National Institutes of Health grants EY08538 and GM34107 (E. Rodriguez-Boulan); EY027038 (R.F. Mullins); 1R21CA224391-01A1 (J.H. Zippin); and 1R01CA194547, 1U24CA210989, and P50CA211024 (O. Elemento); National Cancer Institute grant R01CA192176 and cancer center support grant P30 CA008748-48 (A.L. Joyner); Comunidad Autónoma de Madrid grant 2017-T1/BMD-5247 (I. Benedicto); Agencia Nacional Argentina de Promoción Cient´ıfica y Tecnológica grant PICT 2014-3687 and Fundación Sales (G.A. Rabinovich); a Pew Latin American Fellowship (G.L. Lehmann); Calder Research Scholar Award Vitiligo/Pigment Cell Disorders (J.H. Zippin); Starr Foundation Tri-Institutional Stem Cell Initiative award 2013-028; NYSTEM contract C32596GG; and Research to Prevent Blindness and Dyson Foundation departmental grants. The CNIC is supported by the Instituto de Salud Carlos III, the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Identification and Characterization of Lbh, a Novel Conserved Nuclear Protein Expressed during Early Limb and Heart Development

We report the cloning, protein characterization, and expression of a novel vertebrate gene, termed Lbh (Limb-bud-and-heart), with a spatiotemporal expression pattern that marks embryologically significant domains in the developing limbs and heart. Lbh encodes a highly conserved nuclear protein, which in tissue culture cells possesses a transcriptional activator function. During limb development, expression of Lbh initiates in the ectoderm of the presumptive limb territory in the lateral body wall. As the limb buds appear, Lbh expression is restricted primarily to the distal ventral limb ectoderm and the apical ectodermal ridge, and overlaps in these ectodermal compartments with En1 and Fgf8 expression. During heart formation, Lbh is expressed as early as Nkx2.5 and dHand in the bilateral heart primordia, with the highest levels in the anterior promyocardium. After heart tube fusion and looping, Lbh expression is confined to the ventricular myocardium, with the highest intensity in the right ventricle and atrioventricular canal, as well as in the sinus venosus. Based on the molecular characteristics and the domain-specific expression pattern, it is possible that Lbh functions in synergy with other genes known to be required for heart and limb development

The duration of Fgf8 isthmic organizer expression is key to patterning different tectal-isthmo-cerebellum structures

The isthmic organizer and its key effector molecule, fibroblast growth factor 8 (Fgf8), have been cornerstones in studies of how organizing centers differentially pattern tissues. Studies have implicated different levels of Fgf8 signaling from the mid/hindbrain boundary (isthmus) as being responsible for induction of different structures within the tectal-isthmo-cerebellum region. However, the role of Fgf8 signaling for different durations in patterning tissues has not been studied. To address this, we conditionally ablated Fgf8 in the isthmus and uncovered that prolonged expression of Fgf8 is required for the structures found progressively closer to the isthmus to form. We found that cell death cannot be the main factor accounting for the loss of brain structures near the isthmus, and instead demonstrate that tissue transformation underlies the observed phenotypes. We suggest that the remaining Fgf8 and Fgf17 signaling in our temporal Fgf8 conditional mutants is sufficient to ensure survival of most midbrain/hindbrain cells near the isthmus. One crucial role for sustained Fgf8 function is in repressing Otx2 in the hindbrain, thereby allowing the isthmus and cerebellum to form. A second requirement for sustained Fgf8 signaling is to induce formation of a posterior tectum. Finally, Fgf8 is also required to maintain the borders of expression of a number of key genes involved in tectal-isthmo-cerebellum development. Thus, the duration as well as the strength of Fgf8 signaling is key to patterning of the mid/hindbrain region. By extrapolation, the length of Fgf8 expression could be crucial to Fgf8 function in other embryonic organizers

Altered paracrine signaling from the injured knee joint impairs postnatal long bone growth.

Regulation of organ growth is a poorly understood process. In the long bones, the growth plates (GPs) drive elongation by generating a scaffold progressively replaced by bone. Although studies have focused on intrinsic GP regulation, classic and recent experiments suggest that local signals also modulate GP function. We devised a genetic mouse model to study extrinsic long bone growth modulation, in which injury is specifically induced in the left hindlimb, such that the right hindlimb serves as an internal control. Remarkably, when only mesenchyme cells surrounding postnatal GPs were killed, left bone growth was nevertheless reduced. GP signaling was impaired by altered paracrine signals from the knee joint, including activation of the injury response and, in neonates, dampened IGF1 production. Importantly, only the combined prevention of both responses rescued neonatal growth. Thus, we identified signals from the knee joint that modulate bone growth and could underlie establishment of body proportions

Correct timing of anchoring center initiation is required for correct folial shape

<p><b>Copyright information:</b></p><p>Taken from "Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers"</p><p>http://www.neuraldevelopment.com/content/2/1/26</p><p>Neural Development 2007;2():26-26.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2246128.</p><p></p> Superimposition of midline vermis tracings at E17.5, P0, P3 and adult show that the delay in anchoring center formation of the secondary fissure and premature anchoring center formation of the prepyramidal fissure in mutants result in the a misshapen lobule VIII. Grey arrows demarcate prepyramidal fissure; black arrows demarcate secondary fissure

Purkinje cell layer folding indicates the future positions of the base of each principal fissure

<p><b>Copyright information:</b></p><p>Taken from "Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers"</p><p>http://www.neuraldevelopment.com/content/2/1/26</p><p>Neural Development 2007;2():26-26.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2246128.</p><p></p> () At E16.5 the mouse Cb has a smooth surface, but anti-Calbindin immunostaining (red) shows a multilayer of Pcs with invaginations in the areas where fissures will form (asterisks). Yellow asterisk indicates the fissures shown in (d, e, f). () At E17.5 (b, e) and E18.5 (c, f) both the Pc layer and outer surface invaginate (foliate) simultaneously. Anti-Pax6 immunostaining shows accumulation of the gcps in the EGL, above the Pc layer invagination at E16.5 and E17.5 (inset in (d, e)), whereas by E18.5 the EGL is similar in thickness at the base and at the crown of the folia (inset in (f)). () In animals, is expressed ubiquitously in precursors. Upon administration of tamoxifen at E12.5, ER translocates to the nucleus, where it recombines the floxed STOP signal in the locus. Sagittal sections of E17.5 Cb show that some fate mapped cells (green) colocalize with anti-RORα (red) and are, therefore, Pcs. Marked Pcs (white arrows) have round cell bodies and have extended their axons in various directions. Scale bars: (a-c) 100 μm; (d-f) 40 μm; (g-g2) 15 μm

Gcps of two altered anchoring centers in mutants exhibit the same coordinated changes as in WT

<p><b>Copyright information:</b></p><p>Taken from "Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers"</p><p>http://www.neuraldevelopment.com/content/2/1/26</p><p>Neural Development 2007;2():26-26.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2246128.</p><p></p> Phalloidin staining (red) shows gcp morphology in sagittal sections of P0 WT (a, e) and mutant mice (c, g). At P0, in the WT secondary fissure (a), gcps are more elongated (ci = 0.55) than gcps in the area of the prepyramidal fissure (e) (ci = 0.71). In contrast, in mutants, the gcps in the secondary (c) and prepyramidal (g) fissures are similarly elongated. Dotted white line outlines the EGL, based on the anti-Pax6 immunostaining of adjacent sections to depict the thickness of EGL. Asterisks indicate the base of the fissure. Ci of gcps (gray is WT, white is mutant). Bar height indicates the mean value of each data set, and error bars indicate standard error. An asterisk indicates statistically significant differences between the base of the fissure and crown of the folia for each data set (< 0.0009 for P0; < 0.0002 for P1). Anti-pH3 immunostaining at P0 reveals that there are more gcps in mitosis in the areas where the prepyramidal and secondary fissures will form than at the crown of folium VIII. Bar graphs depict quantification of the number of pH3 positive gcps in the prepyramidal fissure (light blue bars) and in the secondary fissure (dark blue bars) compared to the crown of the intervening folium (red bars). Bar height indicates the mean value of each data set, and error bars indicate standard error. An asterisk indicates statistically significant differences between the two regions (< 0.004 for P0 for the prepyramidal fissure; < 0.05 for P0 for the secondary fissure). Scale bar: 15 μm