Aixa V. Morales

Periodic Lunatic fringe Expression Is Controlled during Segmentation by a Cyclic Transcriptional Enhancer Responsive to Notch Signaling

A molecular oscillator regulates the pace of vertebrate segmentation. Here, we show that the oscillator (clock) controls cyclic initiation of transcription in the unsegmented presomitic mesoderm (PSM). We identify an evolutionarily conserved 2.3 kb region in the murine Lunatic fringe (Lfng) promoter that drives periodic expression in the PSM. This region includes conserved blocks required for enhancing and repressing cyclic Lfng transcription, and to prevent continued expression in formed somites. We also show that dynamic expression in the cycling PSM is lost in the total absence of Notch signaling, and that Notch signaling acts directly via CBF1/RBP-Jkappa binding sites to regulate Lfng. These results are consistent with a model in which oscillatory Notch signaling underlies the segmentation clock and directly activates and indirectly represses Lfng expression.

Role of Prepancreatic (Pro)Insulin and the Insulin Receptor in Prevention of Embryonic Apoptosis

The characterization of (pro)insulin as an early embryonic growth factor requires demonstration of its expression and cellular effects in vivo. By in situ hybridization, we found widespread preproinsulin transcripts in the chick embryo throughout gastrulation and neurulation, before the beginning of preproinsulin-like growth factor I expression and pancreatic organogenesis. To analyze the prepancreatic (pro)insulin effect on apoptotic cell death, we treated embryos with antisense oligodeoxynucleotides in ovo and in vitro. The specific effect of two preproinsulin messenger RNA (mRNA) antisense oligodeoxynucleotides was confirmed by the decrease in a biosynthetically labeled protein immunoprecipitated with antiinsulin Igs. Insulin receptor mRNA antisense oligodeoxynucleotide applied in ovo increased by 2.7-fold the level of apoptosis in the 1.5-day embryo (neurulation) compared with that in its random sequence control. In a whole embryo culture, apoptosis increased by 25-35% with the addition of preproinsulin or insulin receptor mRNAs antisense oligodeoxynucleotides, respectively, whereas it decreased by 64% after 10 h in the presence of 10(-8) M chicken insulin. Exogenous insulin also rescued the death induced by preproinsulin antisense oligonucleotides. These findings provide evidence for an autocrine/paracrine role ofpreproinsulin gene products acting through the insulin receptor in the control of cell survival/death during early embryonic development.

FGF and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk

FGF acts as a positional cue that prevents premature neural crest cell specification and EMT caudally while, at the same time, retinoic acid promotes EMT rostrally.

SOX5 controls cell cycle progression in neural progenitors by interfering with the WNT–β‐catenin pathway

Genes of the SOX family of high‐mobility group transcription factors are essential during nervous system development. In this study, we show that SOX5 is expressed by neural progenitors in the chick spinal cord and is turned off as differentiation proceeds. The overexpression of SOX5 in neural progenitors causes premature cell cycle exit and prevents terminal differentiation. Conversely, knocking down SOX5 protein extends the proliferative period of neural progenitors and causes marked cell death in a dorsal interneuron (dI3) population. Furthermore, SOX5 reduces WNT–β‐catenin signalling, thereby triggering the expression of the negative regulator of the pathway axin2. We propose that SOX5 regulates the timing of cell cycle exit by opposing WNT–β‐catenin activity on cell cycle progression.

Expression of the cCdx-B homeobox gene in chick embryo suggests its participation in rostrocaudal axial patterning.

cCdx‐B (formerly cHox‐cad 2) is a chick homeobox‐containing gene related to the Drosophila caudal. Compared with other caudal homologues, its similarity is highest with the murine Cdx‐4. In the present study, we characterize the localization of cCdx‐B transcripts to the caudal region of the embryo by using reverse transcription‐polymerase chain reaction (RT‐PCR) and, in detail, by using in situ hybridization. Chick embryos from gastrulation to early organogenesis were hybridized with digoxigenin‐labeled riboprobes, and the pattern of expression of cCdx‐B mRNA was analyzed in wholemount embryos and in tissue sections. In the early gastrula, transcripts were localized in a gradient through the caudal half of the embryo, in the epiblast and the mesoderm cells, but not including Hensens node. During neurulation, cCdx‐B transcripts were found more rostrally, with high levels localized in Hensens node and the posterior neural plate. Expression was also high in paraxial mesoderm, with a rostral limit in the most recently formed somite. There was no expression in definitive endoderm. During late neurulation and tail bud formation, cCdx‐B mRNA expression regressed posteriorly and was finally confined to the tail bud region. This pattern of expression of cCdx‐B, regulated in time and space, is different from that of the other known chick caudal homologue, cCdx‐A. Both genes may play a coordinated role in the posterior axial patterning of the chick embryo, whereas cCdx‐B may specify further the identity of the tail region.

Snail genes at the crossroads of symmetric and asymmetric processes in the developing mesoderm.

Retinoic acid (RA) signalling ensures that vertebrate mesoderm segmentation is bilaterally synchronized, and corrects transient interferences from asymmetric left–right (L–R) signals involved in organ lateralization. Snail genes participate in both these processes and, although they are expressed symmetrically in the presomitic mesoderm (PSM), Snail1 transcripts are asymmetrically distributed in the L–R lateral mesoderm. We show that the alteration of the symmetric Snail expression in the PSM induces asynchronous somite formation. Furthermore, in the absence of RA signalling, normal asymmetric Snail1 expression in the lateral mesoderm is extended to the PSM, desynchronizing somitogenesis. Thus, Snail1 is the first cue corrected by RA in the PSM to ensure synchronized bilateral segmentation.

Heat shock proteins in retinal neurogenesis: identification of the PM1 antigen as the chick Hsc70 and its expression in comparison to that of other chaperones

While the role of heat shock proteins under experimental stress conditions is clearly characterized, their expression in unstressed cells and tissues and their functions in normal cell physiology, besides their chaperone action, remain largely undetermined. We report here the identification in chicken of the antigen recognized by the monoclonal antibody PM1 [Hernández‐Sánchez et al. (1994) Eur. J. Neurosci.6,1801–1810) as the non‐inducible chaperone heat‐shock cognate 70 (Hsc70). Its identity was determined by partial peptide sequencing, immuno‐crossreactivity & two‐dimensional gel‐electrophoresis. In addition, we examined its expression during chick embryo retinal neurogenesis. The early widespread Hsc70 immunostaining corresponding to most, if not all, of the neuroepithelial cells becomes restricted to a subpopulation of these cells in the peripheral retina as development proceeds. On the other hand, retinal ganglion cells, differentiating in the opposite central‐to‐peripheral gradient, retained Hsc70 immunostaining. Other molecular chaperones, the heat‐shock proteins Hsp40, Hsp60 & Hsp90, did not seem to compensate the loss of Hsc70. They also showed decreasing immunostaining patterns as neurogenesis proceeds, although distinctive from that of Hsc70, whereas Hsp70 was not detected in the embryonic retina. This precise cellular & developmental regulation of Hsc70, a generally considered constitutive molecular chaperone, in unstressed embryos, together with the expression of other chaperones, provides new tools & a further insight on neural precursor heterogeneity & suggests possible specific cellular roles of chaperone function during vertebrate neurogenesis.

(Pro)insulin and insulin-like growth factor I complementary expression and roles in early development

Evidence that the insulin-like growth factors play a role in embryonic as well as postnatal growth and central nervous system development has accumulated recently from studies using knock-out mice models. However, no effects of IGF-I and II have been demonstrated prior to organogenesis in these studies. We summarize here results supporting the role of insulin (or its precursor proinsulin) in vertebrate development prior to the expression of IGFs. (Pro)insulin mRNA is expressed in the chick embryo during neurulation and early organogenesis and its inhibition by antisense oligodeoxynucleotides increase apoptosis. In another system, proliferative neuroretina, (pro)insulin expression predominates over IGF-I expression. Modulation of apoptosis by (pro)insulin in retina may be largely responsible for the observed stimulation of DNA synthesis and neuronal differentiation. These effects are elicited as well by IGF-I, expressed later in neuroretina. Thus, these polypeptides have complementary expression in early embryos which suggests coordinated actions during development.

Dynamic Sox5 protein expression during cranial ganglia development

Sox5 is a member of the SoxD group of HMG‐box transcription factors that, during the early stages of development, promotes neural crest generation. However, little is known about Sox5 function in neural crest derivatives such as the peripheral sensory nervous system. We have analysed the embryonic expression of Sox5 during chick cranial ganglia development, from the stages of ganglia condensation to those of differentiation. During this period, Sox5 expression is maintained in the crest‐derived satellite glial cells in all the cranial ganglia. In contrast, Sox5 is only transiently expressed in a subpopulation of differentiating neurons of both neural crest and placode origin. This detailed analysis provides a good base to dissect the possible role of Sox5 in neural cell fate determination by future functional approaches. Developmental Dynamics 236:2702–2707, 2007.

Brain Insulin‐Like Growth Factor‐I Directs the Transition from Stem Cells to Mature Neurons During Postnatal/Adult Hippocampal Neurogenesis

The specific actions of insulin‐like growth factor‐I (IGF‐I) and the role of brain‐derived IGF‐I during hippocampal neurogenesis have not been fully defined. To address the influence of IGF‐I on the stages of hippocampal neurogenesis, we studied a postnatal/adult global Igf‐I knockout (KO) mice (Igf‐I−/−) and a nervous system Igf‐I conditional KO (Igf‐IΔ/Δ). In both KO mice we found an accumulation of Tbr2+‐intermediate neuronal progenitors, some of which were displaced in the outer granule cell layer (GCL) and the molecular layer (ML) of the dentate gyrus (DG). Similarly, more ectopic Ki67+‐ cycling cells were detected. Thus, the GCL was disorganized with significant numbers of Prox1+‐granule neurons outside this layer and altered morphology of radial glial cells (RGCs). Dividing progenitors were also generated in greater numbers in clonal hippocampal stem cell (HPSC) cultures from the KO mice. Indeed, higher levels of Hes5 and Ngn2, transcription factors that maintain the stem and progenitor cell state, were expressed in both HPSCs and the GCL‐ML from the Igf‐IΔ/Δ mice. To determine the impact of Igf‐I deletion on neuronal generation in vivo, progenitors in Igf‐I−/− and Igf‐I+/+ mice were labeled with a GFP‐expressing vector. This revealed that in the Igf‐I−/− mice more GFP+‐immature neurons were formed and they had less complex dendritic trees. These findings indicate that local IGF‐I plays critical roles during postnatal/adult hippocampal neurogenesis, regulating the transition from HPSCs and progenitors to mature granule neurons in a cell stage‐dependent manner. Stem Cells 2016;34:2194–2209