Shh Signalling and the Specification of Neuronal Identity

During development, many signalling factors behave as morphogens, long-range signals eliciting different cellular responses according to their concentration. In ventral regions of the spinal cord, Shh is such a signal and controls the emergence, in precise spatial order, of distinct neuronal subtypes. The Gli family of transcription factors play a central role in this process, however, how the graded aspect of Shh signalling controls differential gene expression has been unclear. To address this question we have taken a gain of function approach in chick embryos. We have designed dominant inhibitory and dominant active versions of Gli proteins that are sufficient to block all Gli mediated transcription or mimic positive Gli activity, respectively. Analysis of the effect of these constructs when ectopically expressed in chick embryonic spinal cord indicates that blocking Gli mediated transcription prevents ventral neural tube patterning and the generation of the appropriate neuronal subtypes resulting in the dorsalisation of the ventral neural tube. Conversely, Gli dependent transcription is sufficient to mediate the full range of Shh responses in the neural tube. Moreover, the 2-3 fold changes in Shh concentration, which are necessary to switch between alternative neuronal subtypes in vitro, can be mimicked in vivo by similarly small changes in the level of Gli activity. This analysis also indicated that cells integrate the level of Shh signalling over time suggesting that signal duration in addition to signal strength is an important parameter controlling dorsal-ventral patterning. Together, these data indicate that graded Shh signalling is translated into a gradient of Gli activity without substantial signal amplification and that small changes in the level of Gli activity are sufficient to orchestrate the patterning of the ventral neural tube

Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease

The chicken talpid3 gene encodes a novel protein that is essential for hedgehog signaling

Talpid(3) is a classical chicken mutant with abnormal limb patterning and malformations in other regions of the embryo known to depend on Hedgehog signaling. We combined the ease of manipulating chicken embryos with emerging knowledge of the chicken genome to reveal directly the basis of defective Hedgehog signal transduction in talpid(3) embryos and to identify the talpid(3) gene. We show in several regions of the embryo that the talpid(3) phenotype is completely ligand independent and demonstrate for the first time that talpid(3) is absolutely required for the function of both Gli repressor and activator in the intracellular Hedgehog pathway. We map the talpid(3) locus to chromosome 5 and find a frameshift mutation in a KIAA0586 ortholog (ENSGALG00000012025), a gene not previously attributed with any known function. We show a direct causal link between KIAA0586 and the mutant phenotype by rescue experiments. KIAA0586 encodes a novel protein, apparently specific to vertebrates, that localizes to the cytoplasm. We show that Gli3 processing is abnormal in talpid(3) mutant cells but that Gli3 can still translocate to the nucleus. These results suggest that the talpid(3) protein operates in the cytoplasm to regulate the activity of both Gli repressor and activator proteins

Delta1 Expression, Cell Cycle Exit, and Commitment to a Specific Secretory Fate Coincide within a Few Hours in the Mouse Intestinal Stem Cell System

The stem cells of the small intestine are multipotent: they give rise, via transit-amplifying cell divisions, to large numbers of columnar absorptive cells mixed with much smaller numbers of three different classes of secretory cells - mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, and bactericide-secreting Paneth cells. Notch signaling is known to control commitment to a secretory fate, but why are the secretory cells such a small fraction of the population, and how does the diversity of secretory cell types arise? Using the mouse as our model organism, we find that secretory cells, and only secretory cells, pass through a phase of strong expression of the Notch ligand Delta1 (Dll1). Onset of this Dll1 expression coincides with a block to further cell division and is followed in much less than a cell cycle time by expression of Neurog3 – a marker of enteroendocrine fate – or Gfi1 – a marker of goblet or Paneth cell fate. By conditional knock-out of Dll1, we confirm that Delta-Notch signaling controls secretory commitment through lateral inhibition. We infer that cells stop dividing as they become committed to a secretory fate, while their neighbors continue dividing, explaining the final excess of absorptive over secretory cells. Our data rule out schemes in which cells first become committed to be secretory, and then diversify through subsequent cell divisions. A simple mathematical model shows how, instead, Notch signaling may simultaneously govern the commitment to be secretory and the choice between alternative modes of secretory differentiation

A gene regulatory network balances neural and mesoderm specification during vertebrate trunk development

Transcriptional networks, regulated by extracellular signals, control cell fate decisions and determine the size and composition of developing tissues. One example is the network controlling bipotent neuromesodermal progenitors (NMPs) that fuel embryo elongation by generating spinal cord and trunk mesoderm tissue. Here, we use single-cell transcriptomics to identify the molecular signature of NMPs and reverse engineer the mechanism that regulates their differentiation. Together with genetic perturbations, this reveals a transcriptional network that integrates opposing retinoic acid (RA) and Wnt signals to determine the rate at which cells enter and exit the NMP state. RA, produced by newly generated mesodermal cells, provides feedback that initiates NMP generation and induces neural differentiation, thereby coordinating the production of neural and mesodermal tissue. Together, the data define a regulatory network architecture that balances the generation of different cell types from bipotential progenitors in order to facilitate orderly axis elongation

Μελέτη των αλληλεπιδράσεων μεταξύ των πρωτεϊνών Mox και Pax

Διατμηματικό, συνεργαζόμενα Τμήματα Βιολογίας και Ιατρικής. Mox genes constitute a subfamily of non-clustered, antennapedia-like homeobox genes that are expressed in a wide range of mesodermal tissues and organs. Pax genes are implicated in developmental processes and contain a conserved DNA binding domain, the paired domain. Mox1, Mox2, Pax1 and Pax3 genes are expressed during embryonic development of mouse, human and other organisms. Their expression patterns in the developing somite are not identical but have grate similarities regarding the time and regions of expression. Mox1 null mice and Pax1 mutants show major malformatios in their axial skeleton. Mox2 null mice and Pax3 mutants show defects in the limb myogenesis. Considering that the genes mentioned above are involved in common mechanisms of axial skeleton development (Pax1, Mox1) or formation and differentiation of limb musculature (Pax3, Mox2), we examined if they act in the same developmental pathway through protein-protein interaction. We investigated this hypothesis using three experimental approaches: 1. The yeast two hybrid system, 2. In vitro protein-protein interactions and 3. Coimmunoprecipitation from nuclear extracts of transiently transfected cells. Although the coimmunoprecipitation did not succeed the results from the other two experiments showed that Mox1 can interact with Pax1 and Mox2 with Pax3. The role of these interactions is not known yet.Τα Mox γονίδια (Mox-1 και Mox-2) αποτελούν μια υποοικογένεια homeobox γονιδίων που δεν οργανώνονται σε συστάδες και εκφράζονται σε ιστούς και όργανα μεσοδερμικής προέλευσης. Τα Pax γονίδια εμπλέκονται, όπως και τα Mox, στην εμβρυική ανάπτυξη και περιέχουν μια συντηρημένη δομική περιοχή με ικανότητα πρόσδεσης στο DNA, την paired domain. Τα γονίδια Mox1, Mox2, Pax1 και Pax3 εκφράζονται κατά την εμβρυική ανάπτυξη του ποντικού, του ανθρώπου και άλλων οργανισμών. Η έκφρασή τους εντοπίζεται στους αναπτυσσόμενους σωμίτες και παρουσιάζει επικαλύψεις ως προς το χρόνο και τις περιοχές έκφρασης. Τα ζώα που φέρουν μεταλλαγές αδρανοποίησης είτε του Mox1 είτε του Pax1 παρουσιάζουν αλλοιώσεις στον αξονικό σκελετό. Όσον αφορά τα Mox2 και Pax3 τα μεταλλαγμένα ζώα παρουσιάζουν ανωμαλίες στην ανάπτυξη των μυών στα άκρα. Θεωρώντας ότι τα παραπάνω γονίδια εμπλέκονται σε κοινούς μηχανισμούς διαμόρφωσης του αξονικού σκελετού (Pax1, Mox1) και των σκελετικών μυών του άκρου (Pax3, Mox2) θελήσαμε να εξετάσουμε αν υπάρχει άμεση αλληλεπίδραση μεταξύ των πρωτεϊνών που κωδικοποιούν. Αρχικά με το σύστημα των δυο υβριδίων στον σακχαρομύκητα και στη συνέχεια με in vitro αλληλεπιδράσεις των πρωτεϊνών καταλήξαμε στο συμπέρασμα ότι υπάρχει δυνατότητα αλληλεπίδρασης μεταξύ των Mox1-Pax1 και Mox2-Pax3. Η απόπειρα να συνανοσοκατακρημνίσουμε τις πρωτεϊνες από εκχυλίσματα παροδικά διαμολυσμένων κυττάρων δεν απέδωσε. Ο βιολογικός ρόλος των πρωτεϊνικών αλληλεπιδράσεων που αναφέρουμε παραπάνω δεν είναι γνωστός

Μελέτη των αλληλεπιδράσεων μεταξύ των πρωτεϊνών Mox και Pax

Διατμηματικό, συνεργαζόμενα Τμήματα Βιολογίας και Ιατρικής. Mox genes constitute a subfamily of non-clustered, antennapedia-like homeobox genes that are expressed in a wide range of mesodermal tissues and organs. Pax genes are implicated in developmental processes and contain a conserved DNA binding domain, the paired domain. Mox1, Mox2, Pax1 and Pax3 genes are expressed during embryonic development of mouse, human and other organisms. Their expression patterns in the developing somite are not identical but have grate similarities regarding the time and regions of expression. Mox1 null mice and Pax1 mutants show major malformatios in their axial skeleton. Mox2 null mice and Pax3 mutants show defects in the limb myogenesis. Considering that the genes mentioned above are involved in common mechanisms of axial skeleton development (Pax1, Mox1) or formation and differentiation of limb musculature (Pax3, Mox2), we examined if they act in the same developmental pathway through protein-protein interaction. We investigated this hypothesis using three experimental approaches: 1. The yeast two hybrid system, 2. In vitro protein-protein interactions and 3. Coimmunoprecipitation from nuclear extracts of transiently transfected cells. Although the coimmunoprecipitation did not succeed the results from the other two experiments showed that Mox1 can interact with Pax1 and Mox2 with Pax3. The role of these interactions is not known yet.Τα Mox γονίδια (Mox-1 και Mox-2) αποτελούν μια υποοικογένεια homeobox γονιδίων που δεν οργανώνονται σε συστάδες και εκφράζονται σε ιστούς και όργανα μεσοδερμικής προέλευσης. Τα Pax γονίδια εμπλέκονται, όπως και τα Mox, στην εμβρυική ανάπτυξη και περιέχουν μια συντηρημένη δομική περιοχή με ικανότητα πρόσδεσης στο DNA, την paired domain. Τα γονίδια Mox1, Mox2, Pax1 και Pax3 εκφράζονται κατά την εμβρυική ανάπτυξη του ποντικού, του ανθρώπου και άλλων οργανισμών. Η έκφρασή τους εντοπίζεται στους αναπτυσσόμενους σωμίτες και παρουσιάζει επικαλύψεις ως προς το χρόνο και τις περιοχές έκφρασης. Τα ζώα που φέρουν μεταλλαγές αδρανοποίησης είτε του Mox1 είτε του Pax1 παρουσιάζουν αλλοιώσεις στον αξονικό σκελετό. Όσον αφορά τα Mox2 και Pax3 τα μεταλλαγμένα ζώα παρουσιάζουν ανωμαλίες στην ανάπτυξη των μυών στα άκρα. Θεωρώντας ότι τα παραπάνω γονίδια εμπλέκονται σε κοινούς μηχανισμούς διαμόρφωσης του αξονικού σκελετού (Pax1, Mox1) και των σκελετικών μυών του άκρου (Pax3, Mox2) θελήσαμε να εξετάσουμε αν υπάρχει άμεση αλληλεπίδραση μεταξύ των πρωτεϊνών που κωδικοποιούν. Αρχικά με το σύστημα των δυο υβριδίων στον σακχαρομύκητα και στη συνέχεια με in vitro αλληλεπιδράσεις των πρωτεϊνών καταλήξαμε στο συμπέρασμα ότι υπάρχει δυνατότητα αλληλεπίδρασης μεταξύ των Mox1-Pax1 και Mox2-Pax3. Η απόπειρα να συνανοσοκατακρημνίσουμε τις πρωτεϊνες από εκχυλίσματα παροδικά διαμολυσμένων κυττάρων δεν απέδωσε. Ο βιολογικός ρόλος των πρωτεϊνικών αλληλεπιδράσεων που αναφέρουμε παραπάνω δεν είναι γνωστός

Isolation of the avian homologue of the homeobox gene Mox2 and analysis of its expression pattern in developing somites and limbs

We have isolated the cDNA of avian Mox2 and analyzed its expression pattern during somitogenesis and limb bud formation. Mox2 plays an important role in limb muscle differentiation in the mouse. Mox2 is expressed in the somites of developing chick embryos and in presumptive migrating myoblasts from the dermomyotome to the limb buds. It is also expressed in the ventral and dorsal part of limb buds and is associated with non-proliferating myoblasts. Significant differences were observed in chick and mouse expression patterns, namely in the chick dermomyotome and limb

A gradient of Gli activity mediates graded Sonic Hedgehog signaling in the neural tube

During development, many signaling factors behave as morphogens, long-range signals eliciting different cellular responses according to their concentration. In ventral regions of the spinal cord, Sonic Hedgehog (Shh) is such a signal and controls the emergence, in precise spatial order, of distinct neuronal subtypes. The Gli family of transcription factors plays a central role in this process. Here we demonstrate that a gradient of Gli activity is sufficient to mediate, cell-autonomously, the full range of Shh responses in the neural tube. The incremental two- to threefold changes in Shh concentration, which determine alternative neuronal subtypes, are mimicked by similar small changes in the level of Gli activity, indicating that a gradient of Gli activity represents the intracellular correlate of graded Shh signaling. Moreover, our analysis suggests that cells integrate the level of signaling over time, consistent with the idea that signal duration, in addition to signal strength, is an important parameter controlling dorsal-ventral patterning. Together, these data indicate that Shh signaling is transduced, without amplification, into a gradient of Gli activity that orchestrates patterning of the ventral neural tube

Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity

Sonic hedgehog (Shh) plays a critical role in organizing cell pattern in the developing spinal cord. Gli proteins are thought to mediate Shh signaling, but their role in directing neural tube patterning remains unclear. Here we identify a role for Gli3 transcriptional repressor activity in patterning the intermediate region of the spinal cord that complements the requirement for Gli2 in ventral regions. Moreover, blocking all Gli responses results in a complete dorsalization of ventral spinal cord, indicating that in addition to the specific roles of Gli2 and Gli3 in the neural tube, there is functional redundancy between Gli proteins. Finally, analysis of Shh/Gli3 compound mutant mice substantiates the idea that ventral patterning may involve a mechanism independent, or parallel, to graded Shh signaling. However, even in the absence of graded Shh signaling, Gli3 is required for the dorsal-ventral patterning of the intermediate neural tube. Together these data raise the possibility that Gli proteins act as common mediators integrating Shh signals, and other sources of positional information, to control patterning throughout the ventral neural tube