Engrailed-1 misexpression in chick embryos prevents apical ridge formation but preserves segregation of dorsal and ventral ectodermal compartments

Using lineage tracers, we recently showed dorsal and ventral ectodermal compartments along the sides of the body in chick embryos. The compartments are formed both in presumptive limb-forming regions where they position the apical ridge and also in presumptive interlimb (flank). Here we show, using a novel technique combining fate mapping and in situ hybridisation, that the ventral compartment coincides with the Engrailed-1 (En-1) domain of expression. This coincidence suggests that En-1 could maintain the ventral compartment and be necessary for apical ridge formation. To test this hypothesis, we ectopically expressed En-1 via retroviral transfer and then examined limb development and cell lineage restriction in the ectoderm. En-1 misexpression can completely prevent formation of both normal limbs and ectopic limbs induced in the flank by application of FGF-2. In both cases, there are no morphological signs of apical ectodermal ridge formation and expression of ridge-associated genes is undetectable. In striking contrast, the lineage restriction between dorsal and ventral ectoderm is not altered. Therefore, En-1 is involved in the regulation of ridge formation but not compartment maintenance.     

Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing β-cells

The Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Cre-based fate mapping to indelibly label pancreatic Notch-responsive cells (PNCs) at larval stages and follow their fate in the adult pancreas. We show that the PNCs represent a population of progenitors that can differentiate to multiple lineages, including adult ductal cells, centroacinar cells (CACs) and endocrine cells. These endocrine cells include the insulin-producing β-cells. CACs are a functional component of the exocrine pancreas; however, our fate-mapping results indicate that CACs are more closely related to endocrine cells by lineage as they share a common progenitor. The majority of the exocrine pancreas consists of the secretory acinar cells; however, we only detect a very limited contribution of PNCs to acinar cells. To explain this observation we re-examined early events in pancreas formation. The pancreatic anlage that gives rise to the exocrine pancreas is located in the ventral gut endoderm (called the ventral bud). Ptf1a is a gene required for exocrine pancreas development and is first expressed as the ventral bud forms. We used transgenic marker lines to observe both the domain of cells expressing ptf1a and cells responding to Notch signaling. We do not detect any overlap in expression and demonstrate that the ventral bud consists of two cell populations: a ptf1-expressing domain and a Notch-responsive progenitor core. As pancreas organogenesis continues, the ventral bud derived PNCs align along the duct, remain multipotent and later in development differentiate to form secondary islets, ducts and CACs.     

The CNS midline cells and spitz class genes are required for proper patterning of Drosophila ventral neuroectoderm.

The Drosophila embryonic central nervous system (CNS) develops from sets of neuroblasts (NBs) which segregate from the ventral neuroectoderm during early embryogenesis. It is not well established how each individual NB in the neuroectoderm acquires its characteristic identity along the dorsal-ventral axis. Since it is known that CNS midline cells and spitz class genes (pointed, rhomboid, single-minded, spitz and Star) are required for the proper patterning of ventral CNS and epidermis originated from the ventral neuroectoderm, this study was carried out to determine the functional roles of the CNS midline cells and spitz class genes in the fate determination of ventral NBs and formation of mature neurons and their axon pathways. Several molecular markers for the identified NBs, neurons, and axon pathways were employed to examine marker gene expression profile, cell lineage and axon pathway formation in the spitz class mutants. This analysis showed that the CNS midline cells specified by single-minded gene as well as spitz class genes are required for identity determination of a subset of ventral NBs and for formation of mature neurons and their axon pathways. This study suggests that the CNS midline cells and spitz class genes are necessary for proper patterning of the ventral neuroectoderm along the dorsal-ventral axis.     

Embryogenesis of the murine endocrine pancreas; early expression of pancreatic polypeptide gene.

By immunofluorescence on cytospin preparations and on semithin sections of mouse pancreatic buds, we have found glucagon and pancreatic polypeptide (PP)-containing cells at embryonal day 10.5 (E 10.5) in dorsal buds and at E 11.5 in ventral buds. Insulin-containing cells appear in dorsal buds at E 11.5, and one to two days later in ventral buds. Somatostatin-containing cells are detectable from E 13.5 in both dorsal and ventral buds. A quantitative analysis shows that up to E 15.5, PP-containing cells are relatively abundant in both buds. By PCR amplification of oligo(dT)-primed cDNAs prepared from total pancreatic RNA, we also detect PP mRNA from E 10.5 onwards, thus confirming the early expression of the PP gene in the developing mouse pancreas. Analysis of endocrine cells in situ suggests three major patterns of cell distribution in embryonic pancreas. First, individual hormone-containing cells are located within the epithelium of pancreatic ducts. In both dorsal and ventral buds, the majority of these endocrine cells contain PP, but many also contain glucagon, insulin or somatostatin. Secondly, clusters of endocrine cells are found in the pancreatic interstitium. Many of these cells contain both glucagon and PP which, by immunogold labelling of consecutive thin sections, can be shown to co-exist within individual secretory granules. Finally, starting on E 18.5, typical islets are formed with centrally located B cells and with the adult 'one cell-one hormone' phenotype. These results suggest an intriguing ontogenic relationship between A- and PP-cells, and also indicate that PP-containing cells may occupy a hitherto unexpected place in the lineage of endocrine islet cells.     

Fate map of mouse ventral limb ectoderm and the apical ectodermal ridge

The apical ectodermal ridge (AER) is a critical signaling center at the tip of the limb that promotes outgrowth. In mouse, formation of the AER involves a gradual restriction of AER gene expression from a broad ventral preAER domain to the tip of the limb, as well as progressive thickening of cells to form a multilayered epithelium. The AER is visible from embryonic day 10.5 to 13.5 (E10.5-E13.5) in the mouse forelimb. Previous short-term fate mapping studies indicated that, once a cell is incorporated into the AER, its descendents remain within the AER. In addition, some preAER cells appear to become incorporated into the ventral ectoderm. In the present study, we used an inducible CreER/loxP fate mapping approach in mouse to examine the long-term contribution of preAER cells to limb ventral ectoderm, as well as the ultimate fate of the mature AER cells. We used a CreER transgene that contains Msx2 regulatory sequences specific to the developing AER, and demonstrate by marking preAER cells that, at stage 2 of mouse limb bud development, the majority of the ventral ectoderm that protrudes from the body wall later covers only the paw. Furthermore, when Msx2-CreER-expressing preAER cells are marked after the onset of preAER gene expression, a similar domain of paw ventral ectoderm is marked at E16.5, in addition to the AER. Strikingly, mapping the long-term fate of cells that form the mature AER showed that, although this structure is indeed a distinct compartment, AER-derived cells are gradually lost after E12.5 and no cells remain by birth. A distinct dorsal/ventral border nevertheless is maintained in the ectoderm of the paw, with the distal-most border being located at the edge of the nail bed. These studies have uncovered new aspects of the cellular mechanisms involved in AER formation and in partitioning the ventral ectoderm in mouse limb.     

Differential effects of Drosophila mastermind on asymmetric cell fate specification and neuroblast formation

During neurogenesis in the ventral nerve cord of the Drosophila embryo, Notch signaling participates in the pathway that mediates asymmetric fate specification to daughters of secondary neuronal precursor cells. In the NB4-2 --> GMC-1 --> RP2/sib lineage, a well-studied neuronal lineage in the ventral nerve cord, Notch signaling specifies sib fate to one of the daughter cells of GMC-1. Notch mediates this process via Mastermind (Mam). Loss of function for mam, similar to loss of function for Notch, results in GMC-1 symmetrically dividing to generate two RP2 neurons. Loss of function for mam also results in a severe neurogenic phenotype. In this study, we have undertaken a functional analysis of the Mam protein. We show that while ectopic expression of a truncated Mam protein induces a dominant-negative neurogenic phenotype, it has no effect on asymmetric fate specification. This truncated Mam protein rescues the loss of asymmetric specification phenotype in mam in an allele-specific manner. We also show an interallelic complementation of loss-of-asymmetry defect. Our results suggest that Mam proteins might associate during the asymmetric specification of cell fates and that the N-terminal region of the protein plays a role in this process.     

Normal fate and altered function of the cardiac neural crest cell lineage in retinoic acid receptor mutant embryos

Mouse embryos lacking the retinoic acid (RA) receptors RARalpha1 and RARbeta suffer from a failure to properly septate (divide) the early outflow tract of the heart into distinct aortic and pulmonary channels, a phenotype termed persistent truncus arteriosus. This phenotype is associated with a failure in the development of the cardiac neural crest cell lineage, which normally forms the aorticopulmonary septum. In this study, we examined the fate of the neural crest lineage in RARalpha1/RARbeta mutant embryos by crossing with the Wnt1-cre and conditional R26R alleles, which together constitute a genetic lineage marker for the neural crest. We find that the number, migration, and terminal fate of the cardiac neural crest is normal in mutant embryos; however, the specific function of these cells in forming the aorticopulmonary septum is impaired. We furthermore show that the neural crest cells themselves do not utilize retinoid receptors and do not respond to RA during this process, but rather that the phenotype is cell non-autonomous for the neural crest cell lineage. This suggests that an alternative tissue in the vicinity of the outflow tract of the heart responds directly to RA, and thereby induces or permits the neural crest cell lineage to initiate aorticopulmonary septation.     

Generation of oligodendrocyte precursor cells from mouse dorsal spinal cord independent of Nkx6 regulation and Shh signaling

In the developing spinal cord, early progenitor cells of the oligodendrocyte lineage are induced in the motor neuron progenitor (pMN) domain of the ventral neuroepithelium by the ventral midline signal Sonic hedgehog (Shh). The ventral generation of oligodendrocytes requires Nkx6-regulated expression of the bHLH gene Olig2 in this domain. In the absence of Nkx6 genes or Shh signaling, the initial expression of Olig2 in the pMN domain is completely abolished. In this study, we provide the in vivo evidence for a late phase of Olig gene expression independent of Nkx6 and Shh gene activities and reveal a brief second wave of oligodendrogenesis in the dorsal spinal cord. In addition, we provide genetic evidence that oligodendrogenesis can occur in the absence of hedgehog receptor Smoothened, which is essential for all hedgehog signaling.     

Local extrinsic signals determine muscle and endothelial cell fate and patterning in the vertebrate limb

Both the muscle and endothelium of the vertebrate limb derive from somites. We have used replication-defective retroviral vectors to analyze the lineage relationships of these somite-derived cells in the chick. We find that myogenic precursors in the somites or proximal limb are not committed to forming slow or fast muscle fibers, particular anatomical muscles, or muscles within specific proximal/distal or dorsal/ventral limb regions. Somitic endothelial precursors are uncommitted to forming endothelium in particular proximal/distal or dorsal/ventral limb regions. Surprisingly, we also find that myogenic and endothelial cells are derived from a common somitic precursor. Thus, local extrinsic signals are critical for determining muscle and endothelial patterning as well as cell fate in the limb.     

A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.