Pdm and Castor specify late-born motor neuron identity in the NB7-1 lineage

Abstract

Embryonic development requires generating cell types at the right place (spatial patterning) and the right time (temporal patterning). Drosophila neuroblasts undergo stem cell-like divisions to generate an ordered sequence of neuronal progeny, making them an attractive system to study temporal patterning. Embryonic neuroblasts sequentially express Hunchback, Krüppel, Pdm1/Pdm2 (Pdm), and Castor (Cas) transcription factors. Hunchback and Krüppel specify early-born temporal identity, but the role of Pdm and Cas in specifying temporal identity has never been addressed. Here we show that Pdm and Cas regulate late-born motor neuron identity within the NB7-1 lineage: Pdm specifies fourth-born U4 motor neuron identity, while Pdm/Cas together specify fifth-born U5 motor neuron identity. We conclude that Pdm and Cas specify late-born neuronal identity; that Pdm and Cas act combinatorially to specify a temporal identity distinct from either protein alone, and that Cas repression of pdm expression regulates the generation of neuronal diversity.

Figure 1.

Pdm is required to generate late-born U4 and U5 neurons. (A) Schematic of the wild-type NB7-1 lineage and temporal identity gene expression profile (Schmid et al. 1999; Isshiki et al. 2001; Pearson and Doe 2003). Even-skipped (Eve) is detected in the first five GMCs and their progeny U1–U5 neurons, but not in their neuronal siblings or in the later-born GMCs and interneuronal progeny. Pdm is only transiently detected in U4. Embryonic stages (stage) according to Campos-Ortega and Hartenstein (1997). In this figure and below, neuroblast color reflect the neuronal identity produced at each point in the lineage. We infer that the first interneurons in the lineage arise from a Cas-positive Pdm-negative neuroblast based on the very transient window of Pdm/Cas coexpression, but the identification of markers for these interneurons will be necessary to resolve this point. (B) Schematic of NB7-1 gene expression profile in pdm mutants, based on the timing of gene expression in the entire neuroblast population at the indicated embryonic stages. (C,D) Wild-type and pdm mutant embryos showing Pdm and Cas expression in the neuroblast layer at the indicated embryonic stages. Ventral view of two segments; anterior is up. (E,F) Wild-type and pdm mutant embryos assayed for the indicated U1–U5 neuronal identity markers. (E) In wild type, U1–U5 were present. (F) In pdm mutants, U1–U3 were present but U4/ U5 neurons were often absent (74% both absent; n = 163 hemisegments). Each panel shows one hemisegment of a stage 16 CNS as a maximum intensity projection of the optical sections encompassing the U1–U5 neurons; anterior up, midline to left; neurons are shown as insets if they are obscured in the projection. (Hb) Hunchback; (Kr) Krüppel; (Run) Runt; (Cas) Castor. Summary of U1–U5 phenotype shown at right.

Figure 2.

Prolonged Pdm expression can extend the Pdm/Cas coexpression window and generate ectopic U5 neurons (A) Schematic of NB7-1 gene expression profile in embryos with prolonged Pdm expression (prospero-gal4 UAS-HA:pdm2). (B) prospero-gal4 UAS-HA:pdm2 embryos showing Pdm and Cas in the neuroblast layer at the indicated embryonic stages. Ventral view of two segments; anterior is up. (C) prospero-gal4 UASHA:pdm2 embryos assayed for U1–U5 neuronal identities. Wild-type embryos had U1–U5 neurons (Fig. 1E), whereas prospero-gal4 UASHA:pdm2 embryos typically had four extra U5 neurons in the thorax (n = 58; range 2–5) and at most one extra U5 neuron in the abdomen (n = 68; 12% one extra). Panels show one thoracic or abdominal hemisegment at stage 16; anterior up, midline to left; summary of the most common U1–U5 phenotype is shown at right.

Figure 3.

Precocious Pdm expression can repress Kr, activate cas, and generate ectopic U5 neurons at the expense of U3/U4 neurons. (A) Schematic of NB7-1 gene expression profile in embryos with precocious Pdm expression (engrailed-gal4 UAS-HA:pdm2). (B) engrailed-gal4 UAS-HA:pdm2 embryos stained for Kr and HA:Pdm2 (using anti-HA antibody) at the indicated stages. An optical section through the neuroblast layer is shown. The domain of high HA:Pdm2 in the posterior of each segment (brackets) results in strong repression of Kr and weak activation of Cas; the wild-type pattern of Kr and Cas can be seen in the anterior of each segment where there is no HA:Pdm2 misexpression. Ventral view of two segments; anterior is up. (C) engrailed-gal4 UAS-HA:pdm2 embryos assayed for U1–U5 neuronal identities. Wild-type embryos had U1–U5 neurons (Fig. 1E), whereas engrailed-gal4 UAS-HA:pdm2 embryos had a variable loss of U3/U4 neurons and extra U5 neurons (thoracic segments: 5.2 extra, range 4–9 extra, n = 128; abdominal segments 0–1 extra, n = 359). Panels show one hemisegment of a stage 16 CNS; anterior up, midline to left; summary of the most common phenotypes shown at right.

Figure 4.

Castor is required to suppress U4 and promote U5 neuronal identity. (A) Schematic of NB7-1 gene expression profile in cas mutants. (B) cas²⁴ homozygous mutant embryos showing Pdm in the neuroblast layer at the indicated embryonic stages. Note that Pdm persists until stage 15, which is not observed in wild-type embryos (Fig. 1C). Ventral view of two segments; anterior is up. The neuroblast layer dips out of the focal plane at the segment border (*). (C) cas³⁹ homozygous mutant embryos assayed for U1–U5 neuronal identities. Wild-type embryos had U1– U5 neurons (Fig. 1E), whereas cas mutant embryos had ectopic U4 neurons in both thoracic (6.6 extra; n = 20) and abdominal hemisegments (3.5 extra; n = 29). In this experiment, U5 neurons were identified by expression of a lacZ gene at the cas³⁹ locus, which is expressed in the normal cas pattern despite lack of functional Cas protein (see Materials and Methods for details; see Supplementary Fig. 1A for lacZ expression in the wild-type U5 neuron). Panels show one hemisegment of a stage 16 CNS; anterior up, midline to left; summary of the most common U1–U5 phenotype shown at right. (D) Schematic of NB7-1 gene expression profile in pdm cas²⁴ double mutants. (E) pdm cas²⁴ double-mutant embryos assayed for U1–U5 neuronal identities. Panels show one abdominal hemisegment of a stage 16 CNS; anterior up, midline to left; summary of U1–U5 phenotype shown at right.

Figure 5.

Pdm is required for ectopic U4 neurons in cas mutant embryos. (A) Schematic of NB7-1 gene expression profile in embryos with precocious cas expression (engrailed-gal4 UAS-cas). (B) engrailed-gal4 UAS-cas embryos showing Pdm and Cas in the neuroblast layer at the indicated embryonic stages. Ventral view of two segments; anterior is up. Single channels are shown in rows 1 and 2, which then merge in row 3. Domain of ectopic Cas indicated by brackets. Pdm is down-regulated in the neuroblasts that express ectopic Cas (cf. Fig. 1C). Cas expression can significantly repress Pdm protein levels (quantified in Supplementary Table 1). (C) engrailed-gal4 UAS-cas embryos assayed for U1– U5 neuronal identities. In wild type, we observe U1–U5 neurons (Fig. 1E). In engrailed-gal4 UAS-cas embryos, the U1–U3 fates are normal, but U4 and U5 neurons are invariably missing. Panels show one abdominal hemisegment of a stage 16 CNS; anterior up, midline to left; summary of U1–U5 phenotype shown at right.

Figure 6.

Pdm/Cas specify U5 neuronal identity. (A) Schematic of NB7-1 gene expression profile in embryos with Pdm/Cas misexpression (engrailed-gal4 UASHA:pdm2 UAS-cas). (B) engrailed-gal4 UAS-HA:pdm2 UAS-cas embryos showing Kr, Pdm, and Cas in the neuroblast layer at the indicated embryonic stages. Ventral view of two segments; anterior is up. Kr is repressed in the Pdm/Cas misexpression domain. (C) engrailed-gal4 UASHA:pdm2 UAS-cas embryos assayed for U1–U5 neuronal identities. In wild type, we observe U1–U5 neurons (Fig. 1E). In engrailed-gal4 UAS-HA:pdm2 UAS-cas embryos, we observe loss of Kr+ U3 neurons (79% lost; n = 110) and ectopic U5 neurons (approximately six in thoracic hemisegments, n = 35; ~2.5 in abdominal hemisegments, n = 75). We suggest these extra neurons have a U5 fate based on their Pdm⁻ Run⁺ Cas⁺ marker expression, but we cannot rule out a U4 identity or a mixed U4/U5 identity without additional markers. Panels show one hemisegment of a stage 16 CNS; anterior up, midline to left; summary of U1–U5 phenotype shown at right.

Figure 7.

Regulation of hb, Kr, pdm, and cas neuroblast gene expression. (A) Regulatory hierarchy based on loss-of-function experiments (Isshiki et al. 2001; Kanai et al. 2005; this study). The small Kr below Hb represents low-level Kr that is always coexpressed with Hb; in contrast, the large Kr to the right of Hb represents the high-level Kr expression that occurs after Hb expression is down-regulated. (B) Regulatory hierarchy based on misexpression experiments. In both panels, arrows indicate positive regulation, “T” indicates negative regulation, and “?” indicates one or more unknown transcriptional activators.