Background: Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. In vertebrates, these mechanisms generate "cardinal classes" of neurons that share a transcription factor identity and common morphology. In Drosophila, two cardinal classes are Even-skipped (Eve) + motor neurons projecting to dorsal longitudinal muscles, and Nkx6 + motor neurons projecting to ventral oblique muscles. Cross-repressive interactions prevent stable double-positive motor neurons. The Drosophila neuroblast 7-1 (NB7-1) lineage uses a temporal transcription factor cascade to generate five distinct Eve + motor neurons; the origin and development of Nkx6 + motor neurons remains unclear. Methods: We use a neuroblast specific Gal4 line, sparse labelling and molecular markers to identify an Nkx6 + VO motor neuron produced by the NB7-1 lineage. We use lineage analysis to birthdate the VO motor neuron to the Kr + Pdm + neuroblast temporal identity window. We use gain-and loss-of-function strategies to test the role of Kr + Pdm + temporal identity and the Nkx6 transcription factor in specifying VO neuron identity. Results: Lineage analysis identifies an Nkx6 + neuron born from the Kr + Pdm + temporal identity window in the NB7-1 lineage, resulting in alternation of cardinal motor neuron subtypes within this lineage (Eve>Nkx6 > Eve). Cooverexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineagethe first evidence that this TTF combination specifies neuronal identity. Moreover, the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for motor neuron targeting to ventral oblique muscles, thereby revealing a molecular specification pathway from temporal patterning to cardinal transcription factor expression to motor neuron target selection. Conclusions: We show that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/ Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm > Nkx6 pathway is necessary and sufficient to promote VO motor neuron targeting to the correct ventral muscle group.

11Spatial patterning specifies neural progenitor identity, with further diversity generated by 12 temporal patterning within individual progenitor lineages. These mechanisms generate cardinal 13 classes of motor neurons (sharing a transcription factor identity and common muscle group 14 targets). In Drosophila, two cardinal classes are Even-skipped (Eve)+ motor neurons projecting 15 to dorsal muscles and Nkx6+ motor neurons projecting to ventral muscles. The Drosophila 16 neuroblast 7-1 (NB7-1) lineage generates distinct Eve+ motor neurons via the temporal 17 transcription factor (TTF) cascade Hunchback (Hb)-Krüppel (Kr)-Pdm-Castor (Cas). Here we 18 show that a newly discovered Kr/Pdm temporal identity window gives rise to an Nkx6+ Eve-19 motor neuron projecting to ventral oblique muscles, resulting in alternation of cardinal motor 20 neuron subtypes from a single progenitor (Eve>Nkx6>Eve). We show that co-overexpression of 21 Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage and that Kr/Pdm act via 22 Nkx6, which itself is necessary and sufficient for VO motor neuron identity. Lastly, Nkx6 is 23 required for ventral oblique muscle targeting, thereby linking temporal patterning to motor 24 neuron morphology and synaptic target selection. In conclusion, we show that one neuroblast 25 lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel 26 temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm>Nkx6 27 pathway is necessary and sufficient to specify VO motor neuron identity and morphology. 28 29 Introduction 30 31Neural diversity from flies to mice arises from two major developmental mechanisms. First, neural 32 progenitors acquire a unique and heritable spatial identity based on their position along the 33 rostrocaudal or dorsoventral body axes (Kohwi and Doe, 2013; Sagner and Briscoe, 2019). 34Second, temporal patterning based on neuronal birth-order results in individual progenitors 35 producing a diverse array of neurons and glia (Holguera and Desplan, 2018; Kohwi and Doe, 2013; 36 Miyares and Lee, 2019). Temporal patterning is best characterized in Drosophila; neural 37 progenitors (neuroblasts) located in the ventral nerve cord, central brain, and optic lobes all 38 undergo temporal patterning, in which the neuroblast sequentially expresses a cascade of TTFs 39 that specify distinct neuronal identities (Allan and Thor, 2015; Doe, 2017; Holguera and Desplan, 40 2018; Miyares and Lee, 2019). Although all neuroblasts undergo temporal patterning, the TTFs are 41 different in each region of the brain (Allan and Thor, 2015; Doe, 2017; Holguera and Desplan, 2018; 42 2Miyares and Lee, 2019). Similar mechanisms are used in the mammalian cortex, retina, and spinal 43 cord, although many TTFs remain to be identified (Alsiö et al., 2013; Delile et al., 2019; Elliott et al., 44 2008; Mattar et al., 2015; Sockanathan and Jessell, 1998; Stam et al., 2011). 45A major open question is how transient expression of TTFs like Kr and ...

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. The major substrate of CRL3-KLHL4 is the canonical CDC42 effector kinase PAK1 that monoubiquitylation switches into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific, ubiquitin-dependent restriction of CDC42 signaling is essential for face, brain, and skin formation, demonstrating how cell-fate and morphometric changes are coordinated for faithful organ development.