The prospero gene encodes a divergent homeodomain protein that controls neuronal identity in Drosophila

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The prospero gene encodes a divergent homeodomain protein that controls neuronal identity in Drosophila
Development Supplement 2, 1991, 79-85                                                                                             79
Printed in Great Britain © The Company of Biologists Limited 1991

The prospero gene encodes a divergent homeodomain protein that
controls neuronal identity in Drosophila

QUYNH CHU-LAGRAFF, DOROTHY M. WRIGHT, LESLIE KLIS McNEIL and CHRIS Q. DOE
Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois 61801, USA

Summary

The Drosophila central nervous system (CNS) develops                     in the specification of neuronal fate (Doe et al. 1991).
from a population of stem cells called neuroblasts; each                 Here we show that the pros gene encodes a highly
neuroblast goes through an invariant cell lineage to                     divergent homeodomain. The homeodomain contains
produce a characteristic family of neurons or glia. We                   several of the most conserved amino acids characteristic
are interested in the molecular mechanisms controlling                   of known homeodomains, yet it is considerably less basic
neuroblast cell lineage. Recently we identified the                      than previously identified homeodomains. These data
prospero (pros) gene, which is expressed in embryonic                    are consistent with a model in which pros controls
neuroblasts. Loss of pros function results in aberrant                   neuroblast cell lineages by regulating gene expression.
expression of the homeobox genes fushi tarazu, even-
skipped and engrailed in a subset of neuroblast progeny,                 Key words: Drosophila, CNS, neuroblast, fate, lineage,
suggesting that pros plays an early and fundamental role                 homeodomain.

Introduction                                                             prospero (pros), a neuroblast identity gene (Doe et al.
                                                                         1991). pros is expressed in a subset of neuroblasts and
We are interested in the molecular mechanisms                            newly born GMCs, but not in mature neurons.
controlling cell fate in the Drosophila central nervous                  Embryos lacking pros function (pros embryos) have the
system (CNS). Neurogenesis begins in the ventral                         normal number of neuroblasts, but the neuroblasts
neurogenic region as 25 cells per hemisegment delami-                    generate aberrant cell lineages. In particular, ex-
nate into the embryo to form a subepidermal array of                     pression of the homeobox genes fushi tarazu (ftz), even-
neuronal precursor cells (called neuroblasts); the                       skipped (eve) and engrailed (en) is defective in a subset
remaining superficial cells differentiate into epidermis                 of GMCs, and their neuronal progeny show striking
(Doe et al. 1988a; Jimenez and Campos-Ortega, 1990).                     alterations in axon morphology. In this paper we review
Subsequently, this two-dimensional neuroblast layer is                   the expression and function of pros in developing CNS
transformed into a three-dimensional CNS as each                         and peripheral nervous system (PNS), and describe the
neuroblast goes through an invariant stem cell lineage,                  initial molecular characterization of the pros gene.
'budding off smaller ganglion mother cells (GMCs)
into the embryo. Each GMC divides to produce a
characteristic pair of neurons (Thomas et al. 1984).
   We have proposed that there are three classes of                      Results and discussion
genes controlling cell fate in the developing CNS
(Fig. 1) (Doe et al. 1991; Doe, 1992). First, regionally                 prospero controls the specification of cell fate in the
expressed genes provide positional cues within the                       CNS
neuroectoderm. Second, in response to these positional                   To examine the role of pros in the developing CNS we
cues, 'neuroblast identity' genes are expressed in                       used a variety of cell-specific markers to identify
overlapping subsets of neuroblasts and their progeny.                    individual neuroblasts, GMCs, and neurons in wild-
Each neuroblast expresses a specific combination of                      type and pros embryos (Fig. 2). pros embryos show no
these genes, which leads to the initiation of a unique,                  obvious change in the position or number of neuro-
invariant neuroblast cell lineage. Third, 'GMC identity'                 blasts per segment. However, there are striking defects
genes are expressed in subsets of GMCs in response to                    in neuroblast cell lineages, as detected by altered
the neuroblast identity genes; these genes control the                   expression of cell-specific markers in a subset of
identity of individual GMCs. Examples of 'positional                     neuroblast progeny.
cue' and 'GMC identity' genes are known (Patel et al.                      The earliest defect in pros embryos is a change in
1989; Doe et al. 1988a,b). We recently identified                        GMC gene expression. Normally ftz is expressed in
80     Q. Chu-LaGraff and others

                                                            POSITIONAL              position-specific
                                                            CUES                    neuroblast specification

                                                            NEUROBLAST              translate positional cues
                                                            IDENTITY                into neuroblast cell lineage
                                                            GENES

                                                            GMC
                                                                                    control GMC and
                                                            IDENTITY                neuronal specification
                                                            GENES

Fig. 1. A model for a gene regulatory hierarchy controlling the specification of cell fate in the embryonic CNS. Top:
regionally expressed genes (light and dark stipple) are expressed in overlapping patterns in the ectoderm of the neurogenic
region, such that clusters of 4-6 cells express a unique combination or concentration of gene products (black circles).
Middle: Neuroblast identity genes are expressed in subsets of neuroblasts in response to the positional cues (diagonal
hatching). Each neuroblast identity gene may be expressed in a subset of GMCs produced from a neuroblast; the pattern
of GMC expression can be in lineally related GMCs, positionally related GMCs, or lineage-specific GMCs. The function of
the neuroblast identity genes is to translate positional cues into a specific neuroblast lineage, i.e. the number of cell cycles
and the fate of the GMCs produced. Bottom: GMC identity genes (horizontal hatching) are expressed in subsets of GMCs,
but not neuroblasts, in response to the particular combination of neuroblast identity genes present in a GMC. These genes
are required for the establishment of unique GMC and neuronal fates. Examples are ftz and eve (Doe et al. I988a,b).

about 20 GMCs per hemisegment (which generate 40                  PNS, as well as the individual axon morphology of the
neurons, including the identified aCC and pCC neur-                aCC and pCC neurons. The wild-type CNS has
ons). In pros embryos ftz is expressed in 10-15 GMCs              bilaterally paired longitudinal connectives and two
and about 20-30 neurons; it is not expressed in the aCC           commissural tracts that cross the midline in every
and pCC neurons (Fig. 2). Expression of eve is also               segment, and in each hemisegment there are two nerves
abnormal in pros embryos. In wild-type embryos eve is             connecting the CNS and PNS (Fig. 3). In pros embryos,
expressed in 10 GMCs per hemisegment, which                       the longitudinal connectives fail to form, and the
produce 19 eve-positive neurons, including the aCC and            commissures are reduced and fused (Fig. 3) (Doe et al.
pCC neurons. In pros embryos only 5 GMCs (and 10                   1991).
neurons) express eve; these do not include the aCC and               To determine how individual neurons develop in pros
pCC neurons (Fig. 2). Mature aCC and pCC neurons                  embryos, the aCC and pCC neurons were injected with
can be identified in pros embryos, proving that these             a fluorescent dye. This experiment also serves to verify
neurons do not die in pros embryos; we don't know how             the differentiation of these neurons despite their lack of
many other ftz~ and eve~ neurons also differentiate.              ftz and eve expression. The aCC cell normally extends
Embryos lacking pros show either reduced or ectopic               its axon ipsilaterally into the PNS, pioneering the
expression of GMC markers: ftz and eve show reduced
                                                                  intersegmental nerve; the pCC axon normally grows
expression, whereas en shows ectopic expression
                                                                  anteriorly in a medial fascicle of the longitudinal
(Fig. 2). Thus, pros is required for the correct cell
                                                                  connective (Fig. 3). In pros embryos the 'aCC can be
lineage of several neuroblasts, including the identified
                                                                  identified by its relatively large size and position as the
neuroblast that produces the aCC and pCC neurons.
                                                                  dorsal-most neuron just posterior to the lateral commis-
                                                                  sures. This 'aCC neuron has an abnormal and variable
                                                                  axon morphology. The 'pCC neuron in pros embryos
prospero embryos have axon pathfinding defects in the             has more consistent morphology: it grows anteriorly in
CNS and PNS                                                       a medial fascicle, as in wild-type, and in older embryos
It is known that absence of ftz and eve expression in             it turns medially and crosses the midline (Fig. 3). These
identified GMCs results in abnormal axon pathfinding              results show that the aCC and pCC neurons are born
by their neuronal progeny (Doe et al. 1988a,b). We                and differentiate despite loss of pros, eve, and ftz
assayed the general axon morphology of the CNS and                expression; they do, however, have an abnormal axon
Specification of neuronal precursor cell fates          81
                                                                                             Fig. 2. Loss of pros function
                                                                                             leads to aberrant gene
                                                                                             expression in GMCs and
                                                                                             neurons. (A,B) ftz expression
                                                                                             in wild-type and pros embryos
                                                                                             respectively. (A) Lateral view
                                                                                             of ftz expression in a wild-type
                                                                                             embryo at approx. 7.5 h of
                                                                                             development showing the aCC
                                                                                             and pCC neurons (arrow).
                                                                                             (B) aCC and pCC neurons
                                                                                             (arrow) do not express ftz in
                                                                                             pros embryos. Lateral view,
                                                                                             anterior is up. (C,D) eve
                                                                                             expression in wild-type and
                                                                                             pros embryos respectively.
                                                                                             (C) Ventral view of eve
                                                                                             expression in 13 h wild-type
                                                                                             CNS showing cluster of 10 EL
                                                                                             neurons (arrowhead) and
                                                                                             medial CQ neurons (arrow).
                                                                                             (D) Same view depicting
                                                                                             altered eve expression in 13 h
                                                                                             pros CNS. Note the lack of
                                                                                             eve-positive CQ neurons
                                                                                             (arrow), but normal expression
                                                                                             of EL neurons (arrowhead).
                                                                                             Occasionally there are a few
                                                                                             CQ neurons expressing eve
                                                                                             (asterisk-arrow). (E,F) en
                                                                                             expression in wild type and
                                                                                             pros embryos respectively.
                                                                                             (E) Dorsal view of en
                                                                                             expression in a wild-type 13 h
                                                                                             CNS showing L neurons (outer
                                                                                             arrows), ML neurons (inner
                                                                                             arrows), and the DM cells
                                                                                             (arrowhead). (F) Higher
                                                                                             magnification view of ectopic
                                                                                             en expression in pros CNS
                                                                                             showing en-positive neurons in
the location of the L and ML clusters (arrows), but with additional en-positive neurons in between. There is also an
increase in the number of en-positive DM cells (arrowhead) and absence of large VM en-positive neurons (out of plane of
focus).

morphology. It is unknown whether the aCC and pCC                segmental nerve (Fig. 4; Table 1). In pros embryos the
axon defects are strictly due to loss of pros in their           v and v' axons usually project normally. The d and 1
neuroblast or GMC precursor; some or all of the defects          neurons, however, frequently extend in aberrant
could be due to lack of pros expression in neurons or            directions. The d axons grow in virtually any direction,
glia necessary for proper aCC and pCC axon outgrowth             including dorsally - opposite to their direction of
(Doe et al. 1991). We are currently making embryos               growth in wild-type embryos. Even when the d axons
mosaic for pros CNS expression to resolve this                   extend ventrally, they usually do not fasciculate with
question.                                                        the 1 axons. The 1 neurons also extend axons in a
   Sensory neurons in the PNS of pros embryos show               disoriented fashion, with axon outgrowth ranging from
novel pathfinding defects. Loss of pros function can             ventrally, the normal orientation, to dorsally, which is
result in a reversal of axon polarity, with sensory              never seen in the abdominal segments of wild-type
neurons extending dorsally, away from the CNS                    embryos (Fig. 4; Table 1).
(Fig. 4). In wild-type embryos the neurons and non-                There are several possible explanations for the pros
neuronal support cells of the PNS are arranged in 4              PNS phenotype. First, pros is expressed in many PNS
clusters: ventral (v), ventral' (v'), lateral (1) and dorsal     precursors (see below). Loss of pros expression in
(d) (Ghysen et al. 1986). In wild-type embryos all               neuronal precursors may lead to incorrectly specified
abdominal sensory neuron axons project ventrally into            neurons which are unable to respond to normal
the CNS, with the d and 1 axons fasciculating with the           pathfinding cues. Second, pros is expressed in the
intersegmental nerve and the v' and v axons joining the          mature non-neuronal sheath cell of the lateral chordo-
82       Q. Chu-LaGraff and others

                                              prospero

                                         NBM

           /             \
                                                 \
    •MUIU^.
                     /        \
                                         Q            o
                  aCC         pCC                    "aCC"

J                                   V.
     or
"N

                                                                                     ft*
      aCC                    DCC          "aCC"

     Fig. 3. Summary of pros expression and function in thi
     CNS. Wild type: Neuroblast 1-1 (NB 1-1) and the first
      GMC (GMC-1) express pros (vertical hatching); GMG
      and its progeny (the aCC and pCC neurons) express b<
     ftz and eve (horizontal hatching). Figure directly below
     shows axon of wild-type aCC neuron extending out to
     PNS, and pCC growing anteriorly, prospero: Loss of pi
      expression in GMC 1-1 results in the loss of ftz and evi
      expression and the abnormal development of aCC and
      pCC neurons ('aCC and 'pC') and consequently, loss c
      proper axon pathfinding (figure shown below). The
      drawings are schematics of all the observed axon
      morphologies.
                                                                   Fig. 4. prospero is required for correct PNS axon
                                                                   pathfinding. 14 h embryos showing a lateral view of the
      tonal sensory organ, and in single non-neuronal cells in     regular array of sensory neurons visualized with the SOXII
      other sense organs (Doe et al. 1991). The absence of         antibody; dorsal is up and anterior to the left. (A) Wild-
      pros function from these support cells may affect the        type embryo: the dorsal (d) and lateral (1) neurons extend
      axon projection of the associated neuron. Third, it may      axons ventrally to join with the intersegmental nerve (IS)
      be that the neurons from the 1 and d sense organs            exiting the CNS, while the ventral (v) and ventral' (v')
      require the efferent intersegmental nerve from the CNS       neurons extend axons ventrally in a separate fascicle that
                                                                   joins with the segmental nerve (S) of the CNS. (B) pros
      for correct pathfinding; this nerve is not present in pros   embryo: the d and 1 neurons show striking defects in axon
      embryos (Doe et al. 1991).                                   morphology, often growing dorsally rather than ventrally;
      The prospero gene encodes a predicted protein with a         the intersegmental nerve does not form. The v and v'
                                                                   neurons develop fairly normally and join with the
      highly divergent homeodomain                                 segmental nerve (S) of the CNS.
      An enhancer trap insertion allele (pros139) provided the
      molecular entry point for cloning the pros gene (Doe et
      al. 1991). Two classes of pros cDNAs were isolated:          common with known homeodomains, including the
      prosS and prosL. The prosL predicted protein sequence        four amino acids identical in all higher eukaryotic
      has a number of recognizable motifs: a highly divergent      homeodomains: tryptophan, phenylalanine, aspara-
      homeodomain, several putative nuclear localization           gine, and arginine at positions 49, 50, 52, and 54,
      signals, and basic domains (Wright and Doe, unpub-           respectively (Fig. 5). In addition, the pros homeo-
      lished results). The prosL cDNA is identical to prosS        domain has a predicted helix-turn-helix secondary
      except prosS lacks 87 bp of coding sequence.                 structure resembling helix-turn-helix domains observed
      Interestingly, the missing 87 bp includes part of the        by NMR and crystallography (Kissinger et al. 1990;
      prosL homeobox, resulting in the generation of a             Qian et al. 1989). The pros homeodomains have several
      shorter prosS protein with a different homeodomain           features that are unusual, but not novel. First, most
      (Fig. 5).                                                    homeodomains have 3-4 basic residues at both the N
         Both pros homeodomains have a number of highly            and C termini (Scott et al. 1989); the prosS protein lacks
      conserved and functionally important amino acids in          basic residues entirely from the first and last 7 amino
Specification of neuronal precursor cell fates               83
   Table 1. Axon pathfinding in the prospero PNS.                                      The most highly conserved residues between pros
   Percentage of normal axon connections between                                    and other homeodomains are in the helix 3 region, a
    sensory clusters d, I, v', and v within segments                                domain necessary for sequence-specific DNA binding.
                       T2 to A7                                                     Both NMR and crystal structure studies demonstrate
                                                                                    that helix 3 fits into the major groove of the target
                                         Segments
                                                                                    DNA, with amino acids making contacts with nucleo-
             T2    T3      Al      A2        A3     A4       A5       A6   A7       tides or parts of the phosphate backbone (Kissinger et
                                                                                    al. 1990; Qian et al. 1989). Furthermore, mutation
   d-1       56    38      69       56       56     50       50       56   75
   1-v'      50    50      56       69       56     31       69       81   69       experiments show that residue 9 of helix 3 confers DNA
   v'-v      94    63      88       94       81     81       81       88   69       binding specificity (Hanes and Brent, 1989; Treisman et
                                                                                    al. 1989). The pros homeodomains contain a serine at
   Most axons of the v' cluster successfully fasciculate with the v                 this position, similar to the paired-dass of homeo-
cluster of the PNS. All connections over 80% normal are shown
in bold. Most defects lie in the aberrant axonal growth of the 1
                                                                                    domains (Fig. 5).
clusters and d clusters. n = 16 for each segment.

                                                                                    prospero is expressed in precursors to the CNS and
acids of the homeodomain, and the prosL homeo-                                      PNS
domain has only a single basic residue in these regions                             Due to the nature of pros mutations (aberrant
(Fig. 5). In this regard pros is similar to the yeast mat2-                         neuroblast cell lineages and incorrect GMC specifi-
P homeodomain, which lacks basic residues in both                                   cation), pros expression is expected in a subset of
regions (Kelly et al. 1988), and the labial and hoxl.6                              neuroblasts and GMCs. In fact, pros transcripts are
homeodomains, which have a single N-terminal basic                                  observed in all but two of the neuroblasts in each
residue (Mlodzik et al. 1990; Baron etal. 1987). Second,                            hemisegment (Fig. 6). The two pros-negative cells are
the pros homeodomain is predicted to have 6 amino                                   considered to be neuroblasts, based on morphology and
acids in the turn between helix 2 and 3, whereas most                               position in the neuroblast array. The transcript is clearly
homeodomains have a 3 amino acid turn. However, the                                 restricted to neuroblasts; no signal is seen in the
transcription factor LF-B1 has a 21 residue loop                                    neuroectoderm during the time of neuroblast formation
between helix 2 and 3, and the bacterial LexA protein                               (Fig. 6C). In addition, the transcript is observed in
has a 4 amino acid turn; both of these proteins bind                                many of the GMCs born early in neurogenesis
DNA (Nicosia et al. 1990; Lamerichs et al. 1989; Scott et                           (Fig. 6D). In general, the pros transcript is not
al. 1989).                                                                          detectable in neurons, although it may be expressed in a
                                                                                                               K N N

                                        10                                                                                            50
     piosS        QH APT
     pioaL        L U I S                                                                       Y F p D i IK F | a | T | y « n v K|
     MAI2P        T V R G Q CRU C H K PF¥JM|R|W L Q l H Y D N P|                  N S E F Y D L S A A T G I . T R T F J ] R N|

     pid          K Q R R c R T H F I S A S Q Q D E L E R A Q B R T g|           D I Y T R E E L A Q R T N L T EQR I Q V

     en           D E K R P R|I|A F|S|S E                              E NFJjL    T E R R R Q Q L S S l E L G L,yi
                                                                                                                EJE

     Antp         S R K R G R QQY                                                T R R R R I E I A H A LC 1 T E R

     I-POO         I E It K . . R Q S I A A P E l K R l S    L E A Y Q A Q Q P R S G E       A I A E        D LKKN V V

     CONSERVED               R - - Y          Q          L        1        R                                - L                            I- K - K

     pros a-helice3

     en      a-helices

Fig. 5. Comparison of predicted pros homeodomains with yeast mat2-P, and Drosophila paired iprd), engrailed (en),
Antennapedia (Antp), and I-POU homeodomains (sequences taken from Scott et al. 1989). The prosS homeodomain is
identical to prosL except for the five N-terminal residues. The amino acid similarity between pros and the shown
homeodomains is: mat2-P (15 identities+1 conserved), prd (12+3), en (12+6), Antp (11+3), and I-POU (9+5). The pros
homeodomains share the most amino acid identities with the mat2-P homeodomain, with 28% identity overall and 70%
over 10 amino acids in helix 3. Both pros homeodomains have an extra three amino acids in the turn between helix 2 and
3, which have been excluded to maximize alignment. Black boxes indicate identical amino acids; open boxes indicate
conservative amino acid substitutions. Arrowheads indicate the four amino acids present in all higher eukaryotic
homeodomains. The a-helical domains of the en homeodomain are indicated by an open bar (Kissinger et al. 1990); the
pros (T-helical regions predicted by the Robson-Garnier algorithm are indicated by a black bar.
84     Q. Chu-LaGraff and others

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  This research was supported by the NIH, the Searle             elements of the tripartite DNA binding structure of LFB1. Cell
Scholars Program, and an NSF Presidential Young Investi-         61, 1225-1236.
gator Award.                                                   PATEL, N. H., SCHAFER, B., GOODMAN, C. S. AND HOLMGREN, R.
Fig. 6. prospero transcript is expressed in
                                                                                neuroblasts and GMCs but not in neurons.
                                                                                (A) Enhancer trap detection showing lacZ
                                                                                expression in a pattern matching that of the
                                                                                prospero gene in an approx. 6h embryo (ventral
                                                                                view of about 2.5 segments; compare with panel
                                                                                B). Arrowheads indicate neuroblasts that do not
                                                                                express lacZ. (B) Expression of the pros transcript
                                                                                in most neuroblasts (in an embryo similar in age to
                                                                                that in panel A). The majority of neuroblasts
                                                                                express pros (arrows), but two per hemisegment do
                                                                                not (arrowheads). On the basis of size, morphology
                                                                                and position in the neuroblast array, these two
                                                                                pros-negative cells are neuroblasts; neither is the
                                                                                glioblast. (C,D,E) Neuroblast and GMC transcript
                                                                                expression shown in optical cross-section, detected
                                                                                with HRP reaction product. The ventral surfaces of
                                                                                the embryos are towards the bottom of the photos.
                                                                                (C) pros expression in a newly formed neuroblast
                                                                                (arrow); GMCs have not yet been born. (D) pros
                                                                                expression in newly born GMCs (arrowhead) is
                                                                                often stronger than neuroblast expression (arrow).
                                                                                This photo also illustrates the lack of pros
                                                                                expression in the ventral ectoderm from which the
                                                                                neuroblasts develop (below arrow). (E) Neuroblast
                                                                                expression continues in approx. 7.5 h embryos
                                                                                (arrows), but no neuronal expression (n) is
                                                                                detected; the cell on the dorsal surface expressing
                                                                                pros is one of the longitudinal glia (g). pros is not
                                                                                expressed in the epidermis (e).

B
Fig. 7. pros expression in the PNS. (A) Enhancer trap detection showing lacZ expression the PNS of an approx. 10 h wild-
type embryo. Expression in the sheath cell of the lateral chordotonal sense organs is indicated (segment T3, wide arrow;
segment Al, thin arrow). (B) pros transcript pattern matching the above lacZ pattern in same age embryo; note the
similarity in the chordotonal expression patterns (segment T3, wide arrow; segment Al, thin arrow). Anterior is to the left,
dorsal to the top in both panels.
Specification of neuronal precursor cell fates                   85
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                                                                   with embryonic expression of both prosS and prosL
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