On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

R7 photoreceptor specification requires Notch activity

The eight photoreceptors in each ommatidium of the Drosophila eye are assembled by a process of recruitment [1,2]. First, the R8 cell is singled out, and then subsequent photoreceptors are added in pairs (R2 and R5, R3 and R4, R1 and R6) until the final R7 cell acquires a neuronal fate. R7 development requires the Sevenless receptor tyrosine kinase which is activated by a ligand from R8 [3]. Here, we report that the specification of R7 requires a second signal that activates Notch. We found that a Notch target gene is expressed in R7 shortly after recruitment. When Notch activity was reduced, the cell was misrouted to an R1/R6 fate. Conversely, when activated Notch was present in the R1/R6 cells, it caused them to adopt R7 fates or, occasionally, cone cell fates. In this context, Notch activity appears to act co-operatively, rather than antagonistically, with the receptor tyrosine kinase/Ras pathway in R7 photoreceptor specification. We propose two models: a ratchet model in which Notch would allow cells to remain competent to respond to sequential rounds of Ras signalling, and a combinatorial model in which Notch and Ras signalling would act together to regulate genes that determine cell fate.     

Regulation of R7 and R8 differentiation by the spalt genes

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt and senseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression.     

Analysis of mouse kreisler mutants reveals new roles of hindbrain-derived signals in the establishment of the otic neurogenic domain

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB - a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain - in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3−/−Fgf10−/− double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis.     

cki-1 links cell division and cell fate acquisition in the C. elegans somatic gonad

The formation of a complex multicellular organism requires the precise specification of many diverse cell types at the correct time and position throughout development. This may be achieved by coordinating cell fate specification processes with progression through the cell cycle. Here, we show that the extra distal tip cells (DTCs) associated with the loss of cki-1, a Caenorhabditis elegans homologue of the cyclin-dependent kinase inhibitor p27, do not arise from duplications of pre-existing DTCs, but that they are formed from another cell type within the somatic gonad. Results from our laser microsurgery experiments suggest that the extra DTCs are caused by aberrant somatic gonadal precursor cell divisions in the absence of cki-1, resulting in abnormal daughter cell fates. cki-1(RNAi) animals also possess extra anchor cells and ectopic gonad arms with variable sheath cell numbers and positioning. In addition, cki-1(RNAi) animals display an endomitotic oocyte (Emo) phenotype. Our results uncover a novel role of this CKI in cell fate acquisition, either by directly influencing specification, or through a more conventional role in appropriately linking cell cycle phase with this process.     

Re-programming of C. elegans male epidermal precursor fates by Wnt, Hox, and LIN-12/Notch activities

In Caenorhabditiselegans males, different subsets of ventral epidermal precursor (Pn.p) cells adopt distinct fates in a position-specific manner: three posterior cells, P(9-11).p, comprise the hook sensillum competence group (HCG) with three potential fates (1°, 2°, or 3°), while eight anterior cells, P(1-8).p, fuse with the hyp7 epidermal syncytium. Here we show that activation of the canonical BAR-1 β-catenin pathway of Wnt signaling alters the competence of P(3-8).p and specifies ectopic HCG-like fates. This fate transformation requires the Hox gene mab-5. In addition, misexpression of mab-5 in P(1-8).p is sufficient to establish HCG competence among these cells, as well as to generate ectopic HCG fates in combination with LIN-12 or EGF signaling. While increased Wnt signaling induces predominantly 1° HCG fates, increased LIN-12 or EGF signaling in combination with MAB-5 overexpression promotes 2° HCG fates in anterior Pn.p cells, suggesting distinctive functions of Wnt, LIN-12, and EGF signaling in specification of HCG fates. Lastly, wild-type mab-5 function is necessary for normal P(9-11).p fate specification, indicating that regulation of ectopic HCG fate formation revealed in anterior Pn.p cells reflect mechanisms of pattern formation during normal hook development.     

Embryonic cell lineage of the marine nematode Pellioditis marina

We describe the complete embryonic cell lineage of the marine nematode Pellioditis marina (Rhabditidae) up to somatic muscle contraction, resulting in the formation of 638 cells, of which 67 undergo programmed cell death. In comparison with Caenorhabditis elegans, the overall lineage homology is 95.5%; fate homology, however, is only 76.4%. The majority of the differences in fate homology concern nervous, epidermal, and pharyngeal tissues. Gut and, remarkably, somatic muscle is highly conserved in number and position. Partial lineage data from the slower developing Halicephalobus sp. (Panagrolaimidae) reveal a lineage largely, but not exclusively, built up of monoclonal sublineage blocs with identical fates, unlike the polyclonal fate distribution in C. elegans and P. marina. The fate distribution pattern in a cell lineage could be a compromise between minimizing the number of specification events by monoclonal specification and minimizing the need for migrations by forming the cells close at their final position. The latter could contribute to a faster embryonic development. These results reveal that there is more than one way to build a nematode.     

Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney

The mammalian metanephric kidney is derived from the intermediate mesoderm. In this report, we use molecular fate mapping to demonstrate that the majority of cell types within the metanephric kidney arise from an Osr1⁺ population of metanephric progenitor cells. These include the ureteric epithelium of the collecting duct network, the cap mesenchyme and its nephron epithelia derivatives, the interstitial mesenchyme, vasculature and smooth muscle. Temporal fate mapping shows a progressive restriction of Osr1⁺ cell fates such that at the onset of active nephrogenesis, Osr1 activity is restricted to the Six2⁺ cap mesenchyme nephron progenitors. However, low-level labeling of Osr1⁺ cells suggests that the specification of interstitial mesenchyme and cap mesenchyme progenitors occurs within the Osr1⁺ population prior to the onset of metanephric development. Furthermore, although Osr1⁺ progenitors give rise to much of the kidney, Osr1 function is only essential for the development of the nephron progenitor compartment. These studies provide new insights into the cellular origins of metanephric kidney structures and lend support to a model where Osr1 function is limited to establishing the nephron progenitor pool.     

Eya1 acts upstream of Tbx1, Neurogenin 1, NeuroD and the neurotrophins BDNF and NT-3 during inner ear development

Cell fate specification during inner ear development is dependent upon regional gene expression within the otic vesicle. One of the earliest cell fate determination steps in this system is the specification of neural precursors, and regulators of this process include the Atonal-related basic helix-loop-helix genes, Ngn1 and NeuroD and the T-box gene, Tbx1. In this study we demonstrate that Eya1 signaling is critical to the normal expression patterns of Tbx1, Ngn1, and NeuroD in the developing mouse otocyst. We discuss a potential mechanism for the absence of neural precursors in the Eya1-/- inner ears and the primary and secondary mechanisms for the loss of cochleovestibular ganglion cells in the Eya1bor/bor hypomorphic mutant.     

The anchor cell initiates dorsal lumen formation during C. elegans vulval tubulogenesis

Tubulogenesis and lumen formation are critical to the development of most organs. We study Caenorhabditis elegans vulval and uterine development to probe the complex mechanisms that mediate these events. Development of the vulva and the ventral uterus is coordinated by the inductive cell-signaling activity of a gonadal cell called the anchor cell (AC). We demonstrate that in addition to its function in specifying fate, the AC directly promotes dorsal vulval tubulogenesis. Two types of mutants with defective anchor cell behavior reveal that anchor cell invasion of the vulva is important for forming the toroidal shape of the dorsal vulval cell, vulF. In fos-1 mutants, where the AC cannot breakdown the basement membranes between the gonad and the vulva, and in mutants in unc-6 netrin or its receptor unc-40, which cause AC migration defects, the AC fails to invade the vulva and no lumen is formed in vulF. By examining GFP markers of dorsal vulval cell fate, we demonstrate that fate specification defects do not account for the aberrant vulF shape. We propose that the presence of the AC in the center of the developing vulF toroid is required for dorsal vulval lumen formation to complete vulval tubulogenesis.     

Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest

The Pax3/7 gene family has a fundamental and conserved role during neural crest formation. In people, PAX3 mutation causes Waardenburg syndrome, and murine Pax3 is essential for pigment formation. However, it is unclear exactly how Pax3 functions within the neural crest. Here we show that pax3 is expressed before other pax3/7 members, including duplicated pax3b, pax7 and pax7b genes, early in zebrafish neural crest development. Knockdown of Pax3 protein by antisense morpholino oligonucleotides results in defective fate specification of xanthophores, with complete ablation in the trunk. Other pigment lineages are specified and differentiate. As a consequence of xanthophore loss, expression of pax7, a marker of the xanthophore lineage, is reduced in neural crest. Morpholino knockdown of Pax7 protein shows that Pax7 itself is dispensable for xanthophore fate specification, although yellow pigmentation is reduced. Loss of xanthophores after reduction of Pax3 correlates with a delay in melanoblast differentiation followed by significant increase in melanophores, suggestive of a Pax3-driven fate switch within a chromatophore precursor or stem cell. Analysis of other neural crest derivatives reveals that, in the absence of Pax3, the enteric nervous system is ablated from its inception. Therefore, Pax3 in zebrafish is required for specification of two specific lineages of neural crest, xanthophores and enteric neurons.