Cell autonomous and nonautonomous requirements for Delltalike1 during early mouse retinal neurogenesis

Background: In the vertebrate retina, six neuronal and one glial cell class are produced from a common progenitor pool. During neurogenesis, adjacent retinal cells use Notch signaling to maintain a pool of progenitors by blocking particular cells from differentiating prematurely. In mice there are multiple Notch pathway ligands and receptors, but the role(s) of each paralogue during retinal histogenesis remains only partially defined. Results: Here we analyzed the cell autonomous and nonautonomous requirements for the Deltalike1(Dll1) ligand during prenatal retinogenesis. We used the α‐Cre driver to simultaneously delete a Dll1 conditional allele and activate the Z/EG reporter, then quantified Dll1 mutant phenotypes within and outside of this α‐Cre GFP‐marked lineage. We found that Dll1 activity is required for Hes1 expression, both autonomously and nonautonomously, but were surprised that retinal ganglion cell differentiation is only blocked cell autonomously. Moreover, Dll1 does not act during cone photoreceptor neurogenesis. Finally, Dll1 mutant adult retinas contained small retinal rosettes and RGC patterning defects but were otherwise normal. Conclusions: Although Dll1 participates in bidirectional (cis + trans) Notch signaling to regulate Hes1 expression, it only acts cell autonomously (in cis) to interpret inhibitory signals from other cells that block RGC neurogenesis. Developmental Dynamics 245:631–640, 2016. © 2016 Wiley Periodicals, Inc.     

Differential contribution of Neurog1 and Neurog2 on the formation of cranial ganglia along the anterior-posterior axis

Background: The neural crest (NC) and placode are transient neurogenic cell populations that give rise to cranial ganglia of the vertebrate head. The formation of the anterior NC‐ and placode‐derived ganglia has been shown to depend on the single activity of either Neurog1 or Neurog2. The requirement of the more posterior cranial ganglia on Neurog1 and Neurog2 is unknown. Results: Here we show that the formation of the NC‐derived parasympathetic otic ganglia and placode‐derived visceral sensory petrosal and nodose ganglia are dependent on the redundant activities of Neurog1 and Neurog2. Tamoxifen‐inducible Cre lineage labeling of Neurog1 and Neurog2 show a dynamic spatiotemporal expression profile in both NC and epibranchial placode that correlates with the phenotypes of the Neurog‐mutant embryos. Conclusion: Our data, together with previous studies, suggest that the formation of cranial ganglia along the anterior‐posterior axis is dependent on the dynamic spatiotemporal activities of Neurog1 and/or Neurog2 in both NC and epibranchial placode. Developmental Dynamics 241:229–241, 2012. © 2011 Wiley Periodicals, Inc.     

Cyclin D1 inactivation extends proliferation and alters histogenesis in the postnatal mouse retina

Background: The cell‐cycle regulator Cyclin D1 is expressed in embryonic retinal progenitor cells (RPCs) and regulates their cell‐cycle rate and neurogenic output. We report here that Cyclin D1 also has important functions in postnatal retinal histogenesis. Results: The initial production of Müller glia and bipolar cells was enhanced in Cyclin D1 knockout (Ccnd1^?/?) retinas. Despite a steeper than normal rate of depletion of the RPC population at embryonic ages, postnatal Ccnd1^?/? retinas exhibited an extended window of proliferation, neurogenesis, and gliogenesis. Cyclin D3, normally confined to Müller glia, was prematurely expressed in Ccnd1^?/? RPCs. However, Cyclin D3 did not compensate for Cyclin D1 in regulating cell‐cycle kinetics or neurogenic output. Conclusions: The data presented in this study along with our previous finding that Cyclin D2 was unable to completely compensate for the absence of Cyclin D1 indicate that Cyclin D1 regulates retinal histogenesis in ways not shared by the other D‐cyclins. Developmental Dynamics 241:941–952, 2012. © 2012 Wiley Periodicals, Inc.     

Role of 100S ribosomes in bacterial decay period

Ribosomal proteins S10 and S2 were each fused with GFP to track the fates of these proteins in the stationary growth phase and the following decay period in Escherichia coli. The fused proteins localized mainly in the cytoplasm, and their amounts were proportional to the colony‐forming unit. S10‐GFP strains that lacked genes responsible for regulating 100S ribosomes and S2‐GFP strain that was unable to form 100S both showed shortened stationary phases. This result indicates that these strains exhibit earlier death in the absence of 100S formation (S2‐GFP, S10‐GFP?rmf and S10‐GFP?hpf) and breakdown (S10‐GFP?yfiA). Therefore, in addition to the mere presence of 100S, the correct timing of 100S formation and breakdown is required to maintain viability. We propose a model in which 100S acts as a tentative repository of ribosomes that are protected from degradation and provide a source of amino acids in later growth period.     

Phenotypic diversity and expression of GABAergic inhibitory interneurons during postnatal development in lumbar spinal cord of glutamic acid decarboxylase 67–green fluorescent protein mice

The synthesis enzyme glutamic acid decarboxylase (GAD65 or GAD67) identifies neurons as GABAergic. Recent studies have characterized the physiological properties of spinal cord GABAergic interneurons using lines of GAD67–green fluorescent protein (GFP) transgenic mice. A more complete characterization of their phenotype is required to better understand the role of this population of inhibitory neurons in spinal cord function. Here, we characterize the distribution of lumbar spinal cord GAD67–GFP neurons at postnatal days (P) 0, 7, and 14, and adult based on their co-expression with GABA and determine the molecular phenotype of GAD67–GFP neurons at P14 based on the expression of various neuropeptides, calcium binding proteins, and other markers. At all ages >67% of GFP⁺ neurons were also GABA⁺. With increasing age; (i) GFP⁺ and GABA⁺ cell numbers declined, (ii) ventral horn GFP⁺ and GABA⁺ neurons vanished, and (iii) somatic labeling was reduced while terminal labeling increased. At P14, vasoactive intestinal peptide and bombesin were expressed in ∼63% and ∼35% of GFP⁺ cells, respectively. Somatostatin was found in a small number of neurons, whereas calcitonin gene-related peptide never co-localized with GFP. Moderate co-expression was found for all the Ca²⁺ binding proteins examined. Notably, most laminae I–II parvalbumin⁺ neurons were also GFP⁺. Neurogranin, a protein kinase C substrate, was found in ∼1/2 of GFP⁺ cells. Lastly, while only 7% of GFP⁺ cells contain nitric oxide synthase (NOS), these cells represent a large fraction of all NOS⁺ cells. We conclude that GAD67–GFP neurons represent the majority of spinal GABAergic neurons and that mouse dorsal horn GAD67–GFP⁺ neurons comprise a phenotypically diverse population.     

s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue

Mammary stem cells (MaSCs) play critical roles in normal development and perhaps tumorigenesis of the mammary gland. Using combined cell markers, adult MaSCs have been enriched in a basal cell population, but the exact identity of MaSCs remains unknown. We used the s-SHIP promoter to tag presumptive stem cells with GFP in the embryos of a transgenic mouse model. Here we show, in postnatal mammary gland development, that GFP⁺ cap cells in puberty and basal alveolar bud cells in pregnancy each exhibit self-renewal and regenerative capabilities for all mammary epithelial cells of a new functional mammary gland upon transplantation. Single GFP⁺ cells can regenerate the mammary epithelial network. GFP⁺ mammary epithelial cells are p63⁺, CD24^mod, CD49f^high, and CD29^high; are actively proliferating; and express s-SHIP mRNA. Overall, our results identify the activated MaSC population in vivo at the forefront of rapidly developing terminal end buds (puberty) and alveolar buds (pregnancy) in the mammary gland. In addition, GFP⁺ basal cells are expanded in MMTV-Wnt1 breast tumors but not in ErbB2 tumors. These results enable MaSC in situ identification and isolation via a consistent single parameter using a new mouse model with applications for further analyses of normal and potential cancer stem cells.