The Eya1 Phosphatase Mediates Shh-Driven Symmetric Cell Division of Cerebellar Granule Cell Precursors.

During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates a critical threonine (T410) in the activation loop. Thus, Eya1 inactivates aPKC, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.

Developmental regulation of neuromodulator function in the stomatogastric ganglion of the lobster, Homarus americanus.

Neuromodulatory substances have profound effects on the two motor patterns generated by the adult crustacean stomatogastric ganglion (STG), the gastric mill rhythm and the pyloric rhythm. Developmentally regulated changes in the modulatory functions of neuromodulators could therefore play an important role in the maturation of the output from the developing STG. We compared the effects of neuromodulators on isolated embryonic and adult STG of the lobster, Homarus americanus. Bath application of Val(1)-SIFamide, a peptide whose expression is different in embryos and adults, activated different neuron classes in embryos and adults. Cancer borealis tachykinin-related peptide 1a, a peptide that does not appear in the terminals of modulatory neurons in the STG until after embryonic development, also produced different motor patterns in embryos and adults. In contrast, red pigment concentrating hormone, a peptide with a similar distribution in the STNS across development, produced similar motor patterns in embryonic and adult STG. Proctolin, serotonin, and allatostatin were also physiologically active on the isolated embryonic STG. Together, these results demonstrate that receptors to many neuromodulators are present and functional on STG neurons before the motor patterns of the stomatogastric nervous system are mature. Moreover, neuromodulator responses change during development, perhaps contributing to the maturation of the output from the stomatogastric nervous system.

Spectral analyses reveal the presence of adult-like activity in the embryonic stomatogastric motor patterns of the lobster, Homarus americanus.

The stomatogastric nervous system (STNS) of the embryonic lobster is rhythmically active prior to hatching, before the network is needed for feeding. In the adult lobster, two rhythms are typically observed: the slow gastric mill rhythm and the more rapid pyloric rhythm. In the embryo, rhythmic activity in both embryonic gastric mill and pyloric neurons occurs at a similar frequency, which is slightly slower than the adult pyloric frequency. However, embryonic motor patterns are highly irregular, making traditional burst quantification difficult. Consequently, we used spectral analysis to analyze long stretches of simultaneous recordings from muscles innervated by gastric and pyloric neurons in the embryo. This analysis revealed that embryonic gastric mill neurons intermittently produced pauses and periods of slower activity not seen in the recordings of the output from embryonic pyloric neurons. The slow activity in the embryonic gastric mill neurons increased in response to the exogenous application of Cancer borealis tachykinin-related peptide 1a (CabTRP), a modulatory peptide that appears in the inputs to the stomatogastric ganglion (STG) late in larval development. These results suggest that the STG network can express adult-like rhythmic behavior before fully differentiated adult motor patterns are observed, and that the maturation of the neuromodulatory inputs is likely to play a role in the eventual establishment of the adult motor patterns.

Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster, Homarus americanus.

Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val(1)]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development.

Development of central pattern generating circuits.

The networks that generate rhythmic motor patterns in invertebrates and vertebrates are ideal for studying the mechanisms by which functional circuits are formed during development. Rhythmic motor patterns and movements are seen embryonically, before they are needed for behavior; recent work suggests that activity in immature spinal cord networks is important for circuit formation and transmitter specification. Despite significant advances in describing the patterns of transcription factor expression in both invertebrate nervous systems and vertebrate spinal cord, a real understanding of how central pattern generators develop is hindered by our lack of knowledge of the organization of these circuits in adults.